Objective: To microbiologically evaluate the efficacy of cotton and polytetrafluoroethylene (PTFE) tape used as spacer materials. Method and Materials: Twenty-six extracted human molars were restored using either cotton or PTFE tape as spacers under a standardized provisional restorative material (Cavit). The teeth were incubated for 7 days in a culture of Streptococcus gordonii or in liquid media alone. The spacers were removed and tested for bacterial contamination. The access cavities were also evaluated for bacterial contamination. Results: Nine of 10 teeth with cotton spacers and one of 10 teeth with PTFE spacers were positive for S gordonii growth. The nine teeth in the cotton group also showed contamination of the access cavities. Conclusion: Even under optimal conditions, cotton spacers may cause leakage into the access cavities. Cotton fibers may serve as a route for bacterial contamination of the access cavities and root canal space. In contrast, PTFE tape did not provide an avenue for bacterial contamination. Keywords: cotton, endodontics, PTFE, spacer