Maintaining the sterility of root canal system in between the endodontic treatment appointments is very important to prevent the growth of micro–organism. Aim: To comparatively evaluate the efficacy of cotton and polytetrafluoroethylene (PTFE) tape used as spacer materials under intermediate restoration. Methodology: Twenty patients undergoing root canal treatment in mandibular molar were selected and randomly divided into two groups. After obturation, in the pulp chamber, placed either cotton or PTFE and restored with Glass ionomer restorative material. Samples were obtained from the access cavity at baseline and after seven days. They were checked for microbial contamination with digital colony count. Result: No microbial colony at baseline for both materials, but after seven days, significant increase in microbial growth in both materials. Significant difference in colony count on cotton, compared to PTFE and PTFE showed less microbial contamination. Conclusion: Compared to cotton, PTFE proves to be a better spacer material.