This in vitro study aimed to evaluate the efficacy of the disinfection of root canal systems carried out with Reciproc(TM) and ProTaper Universal(TM) systems using 1% sodium hypochlorite (NaOCl). Forty human single-rooted mandibular premolars were infected with Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus aureus and Candida albicans, and twenty were not infected. The specimens were randomly divided into 6 groups (n = 10): Group 1: ProTaper Universal(TM) + 1% NaOCl; Group 2 (positive control): ProTaper Universal(TM) + saline; Group 3 (negative control without microorganisms): ProTaper Universal(TM) + saline; Group 4: Reciproc(TM) + 1% NaOCl; Group 5 (positive control): Reciproc(TM) + saline; Group 6 (negative control without microorganisms): Reciproc(TM) + saline. Samples were collected before and after the completion of specific treatments, and plated in specific media cultures. The Fisher exact test was used for the statistical analysis of differences in terms of presence or absence of microbial growth among groups. For all tested pathogens, significant differences (p < 0.001) were verified between the instrumentation systems used. ProTaper Universal(TM) associated with 1% NaOCl completely eliminated all microorganisms. Microbial growth, however, was observed when Reciproc(TM) was used associated with 1% NaOCl. According to the protocol executed for this study, the Reciproc(TM) system associated with 1% NaOCl was not able to completely eliminate E. faecalis, P. aeruginosa, S. aureus and C. albicans from the root canal systems.