DNA topoisomerase I (Top1) is a nuclear enzyme that plays a crucial role in the removal of DNA supercoiling associated with replication and transcription. It is also the target of the anticancer agent, camptothecin (CPT). Top1 contains eight cysteines, including two vicinal residues (504 and 505), which are highly conserved across species. In this study, we show that thiol-reactive compounds such as N-ethylmaleimide and phenylarsine oxide can impair Top1 catalytic activity. We demonstrate that in contrast to CPT, which inhibits Top1-catalyzed religation, thiolation of Top1 inhibited the DNA cleavage step of the reaction. This inhibition was more pronounced when Top1 was preincubated with the thiol-reactive compound and could be reversed in the presence of dithiothreitol. We also established that phenylarsine oxide-mediated inhibition of Top1 cleavage involved the two vicinal cysteines 504 and 505, as this effect was suppressed when cysteines were mutated to alanines. Interestingly, mutation of Cys-505 also altered Top1 sensitivity to CPT, even in the context of the double Cys-504 to Cys-505 mutant, which relaxed supercoiled DNA with a comparable efficiency to that of wild-type Top1. This indicates that cysteine 505, which is located in the lower Lip domain of human Top1, is critical for optimal poisoning of the enzyme by CPT and its analogs. Altogether, our results suggest that conserved vicinal cysteines 504 and 505 of human Top1 play a critical role in enzyme catalytic activity and are the target of thiol-reactive compounds, which may be developed as efficient Top1 catalytic inhibitors.