The Reptile Room begins with the three Baudelaire orphans, Violet, Klaus and Sunny, traveling to their new guardian's home along Lousy Lane. Their trip is marred by the strong smell of horseradish and their fears about their new guardian. Dr. Montgomery Montgomery turns out to be much more than the children expected. He seems truly excited to have the children share his home and his work as a herpetologist, or snake expert. He has a job for each of the children, as they get ready for their snake-finding expedition to Peru. The Reptile Room, where Uncle Monty keeps all of his specimens, fascinates the children.

The orphans' joy is short-lived, however, as Uncle Monty's new assistant arrives on the job. His name is Stephano, and despite his lack of hair, shaved eyebrow and beard, the children know that he is really Count Olaf, their evil relative who is after their fortune.

Even though the children desperately try to figure out Count Olaf's plan, he is one step ahead of them. Violet, Klaus and Sunny do their best to warn Uncle Monty of the true identity of Stephano, but their warning comes too late. Uncle Monty is found dead in the Reptile Room, an apparent victim of snakebite. Stephano tries to whisk the children off to Peru, but he crashes into Mr. Poe's car before leaving Uncle Monty's house. The children are delighted to see Mr. Poe, but unfortunately, they cannot convince him that Stephano is really Count Olaf.

Dr. Lucafont arrives to examine the body of Uncle Monty and rules that the death was a result of snakebite. However, the children aren't buying this explanation and go to work to reveal Stephano's guilt and his true identity. Klaus researches the type of snake that Dr. Lucafont claims killed Uncle Monty, and Violet goes to work to find some evidence that will link Stephano to the crime. Through their teamwork, they are able to prove that Uncle Monty's death was not an accident and that Stephano is responsible.

Violet is also able to prove the true identity of Stephano by revealing that he has covered his telltale tattoo with makeup. Once Mr. Poe figures it out, Count Olaf begins to plan his escape. Dr. Lucafont offers to take Count Olaf to the police, but Sunny reveals Lucafont's true identity before they can leave. Dr. Lucafont is the hook-handed man, one of Count Olaf's accomplices.

Just when the children seem to have bested their nemesis, Olaf manages to escape, and Mr. Poe refuses to follow him. Once again, the children will be transferred to a new guardian, always fearing that Count Olaf will be lurking in the shadows, following their every move.