Objectives: the purpose of this study was to analyze the in vitro antimicrobial activity of sodium hypochlorite (1% and 5%) and chlorhexidine (0.12%, 0.5% and 1%) with or without the addition of organic material. Methods: bacterial samples (ATCC) commonly isolated from the root canal system or normally used for antimicrobial tests as Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Porphyromonas gingivalis and Fusobacterium nucleatum were tested by two methods: contact and agar diffusion tests. In the contact test, solutions were analyzed in different intervals of time: immediately (t0), 5 minutes (t5), 15 minutes (t15) and 30 minutes (t30) after contact. The antimicrobial activity was also determinate by the method of agar diffusion. In half of the specimens, 0.5% BSA (bovine serum albumin) was added as organic material. Results: the contact test showed that 0.12% chlorhexidine was not able to eliminate E. faecalis in any interval of time, while 1% chlorhexidine eliminated all strains, and so did both sodium hypochlorite concentrations. The results of the agar diffusion test were statistically analyzed by ANOVA (p=0.00) and Bonferroni t test (p<0.05) that showed statistical difference among the solutions and the chlorhexidine solutions presented the best results when BSA was used. Conclusions: these results permit us to conclude that to obtain a better antimicrobial activity, a chlorhexidine solution in a concentration superior to 0.12% should be used and that the addition of BSA interferes in the solutions activity.