Doxycycline hyclate (DCH) and minocycline hydrochloride (MH) are tetracycline antibiotics and broad-spectrum antimicrobial agents. The changes in DCH and MH under blue light (λ = 462 nm) irradiation in alkaline conditions (BLIA) were investigated. Deactivation caused by superoxide anion radical (O) and deactivation from DCH and MH during photolysis on Staphylococcus aureus (S. aureus), including methicillin-resistant S. aureus (MRSA), were studied. DCH is relatively unstable compared to MH under BLIA. The level of O generated from the MH-treated photoreaction is lower than that from DCH photolysis, and the DCH-treated photoreaction is more efficient at inactivating S. aureus and MRSA at the same radiant intensity. DCH subjected to BLIA decreased the viability of S. aureus and MRSA by 3.84 and 5.15 log, respectively. Two photolytic products of DCH (PPDs) were generated under BLIA. The mass spectra of the PPDs featured molecular ions at m/z 460.8 and 458.8. The molecular formulas of the PPDs were CHNO and CHNO, and their exact masses were 462.44 and 460.44 g/mol, respectively. These results bolster the photolytic oxidation that leads to DCH-enhanced deactivation of S. aureus and MRSA. Photochemical treatment of DCH could be applied as a supplement in hygienic processes.