The aim of this study was to evaluate the antimicrobial capacity of sodium hypochlorite (1% and 5%) and chlorhexidine (0.12%, 0.5% and 1%) with or without the addition of organic material (bovine serum albumin, BSA) against some bacterial samples (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Porphyromonas gingivalis and Fusobacterium nucleatum) using two activity tests (contact and diffusion agar tests). In the contact test (first model), bacterial samples were kept in contact with each irrigating solution for different time intervals: immediately (t(0)), 5 min (t(5)), 15 min (t(15)) and 30 min (t(30)). The agar diffusion test was the second model used. In half the specimens, 0.5% BSA was added to simulate organic tissue present in the root canal. Bacterial growth was evaluated for each microorganism and activity test. Each test was repeated 10 times. In the contact test, 0.12% chlorhexidine solution (CHX) did not eliminate E. faecalis at any tested time. CHX at 0.5% eliminated all strains except E. faecalis after immediate contact. All strains were eliminated by 1% CHX, 1% NaOCl and 5% NaOCl. BSA did not interfere with the antimicrobial activity of the irrigating solutions. In the agar diffusion test, all solutions exhibited zones of antimicrobial activity; however, BSA interfered with the antimicrobial activity of NaOCl and CHX. Under the condition of the contact test, the 0.12% CHX was ineffective in eliminating E. faecalis, while 0.5% CHX, 1% CHX, 1% NaOCl and 5% NaOCl showed antibacterial effectiveness against all the tested bacterial strains. The addition of an organic load interfered with the accuracy of the agar diffusion test.