The aim of this study was to evaluate the effectiveness of ozonated water in the elimination of Candida albicans, Enterococcus faecalis, and endotoxins from root canals. Twenty-four single-rooted human teeth were inoculated with C. albicans and E. faecalis, and 24 specimens were inoculated with Escherichia coli endotoxin. Ozonated water (experimental group) or physiologic solution (control group) was used as irrigant agent. Antimicrobial effectiveness was evaluated by the reduction of microbial counts. Lipopolissacharide complex presence was assessed by limulus amebocyte lysate test and B-lymphocyte stimulation. Data were analyzed by Wilcoxon and Mann-Whitney tests (5%). Ozonated water significantly reduced the number of C. albicans and E. faecalis at the immediate sampling, but increased values were detected after 7 days. Ozonated water did not neutralize endotoxin. It could be concluded that ozonated water was effective against C. albicans and E. faecalis but showed no residual effect. No activity on endotoxin was observed.