The aim of this study was to analyze the effectiveness of 5.25% sodium hypochlorite (NaOCI) in preventing inoculation of periapical tissues with contaminated patency files. Twenty-eight extracted human permanent teeth with single canals were used in the study. Group I teeth were filled with NaOCl, and #15 stainless steel files contaminated with Streptococcus sanguis (ATCC #10556) were allowed to pass through the NaOCI into the culture medium. The teeth in group II were also filled with NaOCl, but the contaminated files used in group II canals were immersed in NaOCl for 10 s prior to being placed into the canals and cultured. The negative control group used sterile files (0% growth), the first positive control group used contaminated patency files in teeth with empty canals (100% growth), and the second positive control group placed contaminated files into broth next to teeth filled with NaOCl (to evaluate potential chlorine leakage; 100% growth). The experimental results showed no positive growth of S. sanguis for groups I and II, indicating that the NaOCl present in the canal after irrigation was sufficient to kill the test organism.