The purpose of the present study was to evaluate the risk of inducing bacterial resistance to disinfection treatment with photolysis of H2O2 and comparing this with existing antibacterial agents. We tested seven antibacterial agents, including amoxicillin, cefepime hydrochloride, erythromycin, ofloxacin, clindamycin hydrochloride, ciprofloxacin hydrochloride, and minocycline hydrochloride, as positive controls for validation of the assay protocol. For all of the agents tested, at least one of the four bacterial species (Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, and Streptococcus salivarius) was resistant to these agents by repeated exposure to subinhibitory concentrations of the agents up to 10 times. In contrast, antibacterial activity against any of the bacterial species tested (S. aureus, E. faecalis, E. coli, S. salivarius, Pseudomonas aeruginosa, Streptococcus mutans, and Aggregatibacter actinomycetemcomitans) was not affected by repeated exposure to the disinfection treatment up to 40 times. This finding suggested that the risk of inducing bacterial resistance by disinfection treatment was low. The active ingredient of this disinfection treatment is hydroxyl radicals generated by photolysis of H2O2. Therefore, hydroxyl radicals interact with several cell structures and different metabolic pathways in microbial cells, probably resulting in a lack of development of bacterial resistance. In conclusion, disinfection treatment with photolysis of H2O2 appears to be a potential alternative for existing antimicrobial agents in terms of a low risk of inducing bacterial resistance.