Background and Objective: Studies on the photobactericidal efficacy of methylene blue (MB) and toluidine blue (TB) have shown inconsistent results in the literature. This study evaluated the bactericidal efficacy of MB and TB against different bacteria under light and dark conditions to determine the most effective bactericidal dye. Study Design/Materials and Methods: Suspensions of Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Hemophilus influenzae, Escherichia coli, and Pseudomonas aeruginosa in saline were treated in dark and red laser light conditions in the presence of each dye using an argon pumped‐dye and a diode laser emitting light at 630 and 664 nm, respectively. The effect of dye concentration, dark incubation time, the fluence and intensity of laser light on the destruction of different bacteria were compared. Results: Both dyes eradicated all examined bacteria under laser light. The complete photodestruction of microorganisms was reached at TB concentrations of 1.5–7‐fold less than that of MB. Conclusion: TB exhibits a greater bactericidal activity than MB against most bacteria in dark and light conditions. Mostly, these results are consistent with their respective dye partition coefficients. Lasers Surg. Med. 29:165–173, 2001. © 2001 Wiley‐Liss, Inc.