The objective of this study was to analyze the antimicrobial effect of 2% sodium hypochlorite (NaOCl) and 2% chlorhexidine (CHX) by agar diffusion test and by direct exposure test. Five microorganisms: Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aernginosa, Bacillus subtilis, Candida albicans, and one mixture of these were used. These strains were inoculated in brain heart infusion (BHI) and incubated at 37 degrees C for 24 h. For the agar diffusion test (ADT), 18 Petri plates with 20 ml of BHI agar were inoculated with 0.1 ml of the microbial suspensions, using sterile swabs that were spread on the medium, obtaining growth injunction. Fifty-four paper disks (9 mm in diameter) were immersed in the experimental solutions for 1 min. Subsequently, three papers disks containing one of the substances were placed on the BHI agar surface in each agar plate. The plates were maintained for 1 h at room temperature, and then incubated at 37 degrees C for 48 h. The diameter of microbial inhibition was measured around the papers disks containing the substances. For the direct exposure test, 162#50 sterile absorbent paper points were immersed in the experimental suspensions for 5 min, and were then placed on Petri plates and covered with one of the irrigant solutions, or with sterile distilled water (control group). After intervals of 5, 1 0 and 30 min, the paper points were removed from contact with the solutions and individually immersed in 7 ml of Letheen Broth, followed by incubation at 37 degrees C for 48 h. Microbial growth was evaluated by turbidity of the culture medium. A 0.1 ml inoculum obtained from the Letheen Broth was transferred to 7 ml of BHI, and incubated at 37 degrees C for 48 h. Bacterial growth was again evaluated by turbidity of the culture medium. Gram stain of BHI cultures was used for verification of contamination and growth was determined by macroscopic and microscopic examination. The best performance of antimicrobial effectiveness of NaOCI was observed in the direct exposure test, and of CHX was observed in the agar diffusion test. The magnitude of antimicrobial effect was influenced by the experimental methods, biological indicators and exposure time.