An antimicrobial technique based on photo-oxidation of caffeic acid (CA) has recently been developed, but its effect on biofilm-forming bacteria is unknown. The present study aimed to evaluate the effect of photo-irradiated CA against Streptococcus mutans (cariogenic bacteria) biofilm as it relates to hydroxyl radical formation. S. mutans biofilms grown on hydroxyapatite disks were immersed in CA solution (0-2 mg/mL) and irradiated with LED light at wavelengths of 365, 385, and 400 nm and at irradiances of 500, 1000, and 2000 mW/cm2 for 4 min. Biofilm viable bacterial counts were determined by colony counting. The yield of hydroxyl radicals generated by the LED irradiation of CA solution was quantified by electron spin resonance analysis. Of the conditions tested, the highest bactericidal effect, with a > 5-log reduction in viable bacterial counts, was obtained by irradiation of a 1 mg/mL CA solution with 385 nm LED and at an irradiance of 2000 mW/cm2. Hydroxyl radical formation was related to this bactericidal effect. The present study suggests that the antimicrobial technique based on the 385 nm LED irradiation of CA is effective against cariogenic biofilms and can be applied as an adjunctive chemotherapy for dental caries.