Objective: Spacers are commonly placed between the canal orifice and the temporary material between endodontic treatment appointments. This prevents the temporary restoration material from obstructing the canal orifices and allows for easy removal. Various endodontic spacers are currently used, including polytetrafluoroethylene (PTFE) tape. Previous in-vitro studies have demonstrated the advantages of using PTFE over using cotton; however, no in-vivo studies have demonstrated this. Hence, the purpose of this study was to evaluate which spacer showed less bacterial leakage between endodontic treatments. Method and Materials: Fifty patients participated in the study and were randomly assigned to either the cotton or the PTFE group. Root canal treatments were completed in two appointments. Cotton and PTFE spacers were collected after a 2- to 4- week time interval between the first and second appointments. Samples were incubated on agar plates for 48 hours and then evaluated for presence of microbial growth. Colony forming units (CFUs) were counted for each of the samples. The results were analyzed using nonparametric statistical tests. Results: Fifteen of the 24 cotton spacers and two of the 24 PTFE spacers were positive for bacterial growth. Conclusion: Cotton fibers exposed to the oral environment could potentially wick contaminants into the pulp chamber. The tendency of cotton to distort under masticatory forces may allow disruption of the temporary material's marginal seal. Based on the results of this study, the use of PTFE is strongly recommended over cotton as an endodontic spacer material. PTFE performed better than cotton in this in-vivo microbial study. Keywords: cotton, endodontic spacers, PTFE, root canal therapy