The purpose of this in vitro study was to analyze the effectiveness of 2.5% sodium hypochlorite (NaOCl) in preventing inoculation of periapical tissue with contaminated patency files. Fifty single-rooted teeth with single canals were used in the study. They were randomly divided into five groups of which two were experimental groups, two positive controls, and one negative control group. After root canal preparation, teeth in Group I (experimental) were filled with 2.5 NaOCl and #15 stainless steel K-files contaminated with Streptococcus sanguis (ATCC# 10556) were allowed to pass through the root canal into the culture medium and cultured. Teeth in Group II (experimental) were also filled with NaOCl, but contaminated files used in this group were immersed in 2.5% NaOCl for 10 s prior to being placed in the canal. The negative control used sterile files pass through 2.5% NaOCl into the culture medium. The first positive control used contaminated patency files in teeth with saline. The second positive control group placed contaminated files into broth next to teeth filled with NaOCl (to evaluate potential chlorine leakage). The results were as follows. Both the experimental groups and the negative control group showed no growth. Both the positive control groups 100% growth for S. sanguis. This indicates that the NaOCl present in the canal after irrigation was sufficient to kill the test organism.