Barchester Towers is the chronicle of personal and political maneuvering within the Church of England in the mid-nineteenth century. The story begins as one Bishop of Barchester dies and a new bishop is selected. This new Bishop, Dr. Proudie, is led by his wife, Mrs. Proudie, and his domestic chaplain, Mr. Slope. Mrs. Proudie and Mr. Slope are determined to shake up the church establishment in Barchester with a series of new policies and practices, including the formation of Sabbath, or Sunday schools. The established clergy of Barchester, led by Archdeacon Grantly, the son of the previous Bishop, are equally determined to keep things as they have always been.

Tangled up in this church power struggle is Hiram's Hospital, a charitable establishment for retired people. A new warden is to be appointed to run this hospital and both sides want to put one of their own followers in this position. The Archdeacon wants to reappoint Dr. Harding, who held the position previously before a newspaper scandal forced him to resign. Dr. Harding is also the father of two daughters. The older daughter is Susan Grantly, who is married to the Archdeacon. The younger is Eleanor Bold, a new widow with a baby and a small fortune.

At first Mrs. Proudie and Mr. Slope are united in their desire to appoint someone of their own group to the position. This unity ends, however, as the two of them fight between themselves for control over the Bishop. At first they work together to get the position for Mr. Quiverful, an extremely poor preacher who has fourteen children and a very small income. Then Mr. Slope realizes that, because of her money, he wants to try to marry Eleanor, and he switches alliances to support Mr. Harding. This begins a struggle between Mrs. Proudie and Mr. Slope to get the Bishop to choose their candidate.

Mr. Slope's interest in Eleanor has begun to worry her family, who think that she might consider marrying Slope, a prospect that fills them with horror. Without consulting Eleanor, they discuss the possible marriage behind her back and decide that if she does marry Slope, they will all refuse to see her anymore. Eleanor, however, has no intention of marrying Slope and no idea that her family thinks she will. She continues to see Mr. Slope because she feels it is wrong to be rude to him just because he has different opinions about the church.

The next complication to enter the story is the return of the Stanhope family, which has been living in Italy the last twelve years. Dr. Stanhope is also a preacher, but he has neglected his post in England in order to live idly in Italy. His children have been raised in Italy and are very badly suited to England. His oldest daughter Charlotte is trying to protect his only son, Bertie, who is lazy, good-natured, and deeply in debt. His other daughter, Madeleine, is crippled from an accident involving her now absent husband, Signor Neroni. She can't walk, is carried everywhere, and her only wish in life is to seduce every man she meets. She refers to herself as "the signora." Soon after their arrival to Barchester, two plots emerge. The first is the signora's seduction of Mr. Slope. The second is Charlotte's plan for Bertie to try to marry Eleanor for her money.

In the ongoing power struggle between the Bishop's group and the Archdeacon, the Archdeacon has decided to call in a respected Oxford scholar, Mr. Arabin, to help him fight for traditional church values in Barchester by giving him the post of preacher at St. Ewold's, a small country church. Mr. Arabin is an older, unmarried, and very honest man who quickly finds himself in love with Eleanor. While Eleanor's family is convinced she will marry Mr. Slope, Eleanor is actually growing closer to Mr. Arabin. Eleanor and her family have a huge fight when the Archdeacon reveals their suspicions, and they refuse to see each other for a while.

Meanwhile, the Bishop has awarded the warden position to Mr. Quiverful, therefore giving Mrs. Proudie a victory and Mr. Slope a defeat. Mr. Slope is not discouraged, however, because a new position as Dean of Barchester Cathedral has just become available. Mr. Slope is determined to gain this new, important post. The Archdeacon's group is equally determined to keep him from getting this position.

At a party to welcome Arabin to his new position at St. Ewold's, both Mr. Slope and Bertie Stanhope propose to Eleanor, which upsets her because she had not realized either of the men thought that she liked them in that way. Also at the party, the signora meets Mr. Arabin and decides, against her usual character, that she will help him to marry Eleanor because she feels he is a good, honest man incapable of being seduced. Two days after the party, the signora meets with Eleanor to tell her that Mr. Arabin loves her. Eleanor loves Mr. Arabin but does not know what she should do about it. This problem is solved when a wealthy neighborhood lady, Miss Thorne, invites both Eleanor and Mr. Arabin to visit her at her country home. When they are left alone together, Mr. Arabin proposes and Eleanor accepts.

Eleanor and Mr. Arabin return to town and announce their engagement. At the same time, Mr. Harding has been informed that he has been chosen to be the new Dean. He turns down the position and gets the church leaders to give it to Mr. Arabin instead. Mr. Slope leaves Barchester in disgrace. Eleanor and Mr. Arabin get married and everyone is very happy for them.