Antimicrobial blue light (aBL) offers efficacy and safety in treating infections. However, the bacterial targets for aBL are still poorly understood and may be dependent on bacterial species. Here, we investigated the biological targets of bacterial killing by aBL (l = 410 nm) on three pathogens: Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Initially, we evaluated the killing kinetics of bacteria exposed to aBL and used this information to calculate the lethal doses (LD) responsible for killing 90 and 99.9% of bacteria. We also quantified endogenous porphyrins and assessed their spatial distribution. We then quantified and suppressed reactive oxygen species (ROS) production in bacteria to investigate their role in bacterial killing by aBL. We also assessed aBL-induced DNA damage, protein carbonylation, lipid peroxidation, and membrane permeability in bacteria. Our data showed that P. aeruginosa was more susceptible to aBL (LD99.9 = 54.7 J/cm2) relative to S. aureus (LD99.9 = 158.9 J/cm2) and E. coli (LD99.9 = 195 J/cm2). P. aeruginosa exhibited the highest concentration of endogenous porphyrins and level of ROS production relative to the other species. However, unlike other species, DNA degradation was not observed in P. aeruginosa. Sublethal doses of blue light (,LD90) could damage the cell membrane in Gram-negative species but not in S. aureus. In all bacteria, oxidative damage to bacterial DNA (except P. aeruginosa), proteins, and lipids occurred after high aBL exposures (.LD99.9). We conclude that the primary targets of aBL depend on the species, which are probably driven by variable antioxidant and DNA-repair mechanisms.Funda????o de Amparo ?? Pesquisa do Estado de S??o Paulo (FAPESP)U.S. Department of DefenseFAPESP: 16/ 25095-2; 19/10851-4U.S. Department of Defense: FA9550-20-1-0063