Objective: The aim of the present study was to evaluate the antimicrobial influence of different root canal filling techniques using gutta-percha and an epoxy resin-based sealer in experimentally infected root canals of extracted human teeth. Method and Materials: In total, 96 intact sterilized, permanent human anterior teeth and premolars with single patent root canals were prepared and infected with a clinical isolate of Enterococcus faecalis. After 72 hours, all root canals were sampled using three sterile paper points. The tooth specimens were randomly divided into three groups and a control of 24 specimens each, according to the respective obturation techniques: lateral condensation (LC group), ProTaper Thermafil (PT group), and vertical compaction technique (VC group). AH Plus was used as sealer. The control group was left untreated (without root canal filling). After 7 days root canal fillings were removed and collected. The root canals were sampled using three sterile paper points and dentin chips were obtained from the root canal walls. The samples were cultured on blood agar, and colony forming units were counted. Results: All root canal filling techniques significantly reduced bacterial viability, eliminating more than 99.9% of E faecalis. In the LC group, three (13%) root canals were culture negative. In the PT group, 21 (88%) root canals and in the VC group 15 (54%) were culture negative. Conclusion: All root canal filling techniques significantly reduced E faecalis in root canals. In cases where warm filling techniques can be applied, these should be preferred to cold obturation. Keywords: bacteria, gutta-percha, lateral compaction, sealer, Thermafil, vertical compaction