Aim: This study aimed to evaluate the sealing ability of cotton and Polytetrafluoroethylene (PTFE) tape as spacer under temporary filling material in between root canal treatment visits . Method and Materials: forty infected primary molars indicated for pulpectomy from twenty children were selected for this study. Two visits root canal treatment were done with temporary restorative material and application of the tested cotton and PTFE as a spacers on each side randomly. Microbiological study was done after pulpectomy by taking sample from the coronal portion of the cavity at the folloing steps, directly after pulpectomy, seven days follow up for both the cavity and the spacer material itself. Results: Cotton fibers group showed bacterial contamination for both the cavity and material itself while PTFE showed the least amount of bacterial growth over their samples. There was contamination of the majority of cotton group samples in contrary to minimal amount for PTFE samples showed bacterial contamination Conclusions: Using PTFE as a spacer material under provisional restoration showed very good results from microbial point of view and it may be used as cotton alternative in the future in the endodontic field