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Biofilms contribute to chronic infections and the development of antimicrobial resistance (AMR). We are developing an antimicrobial blue light (aBL) device to reduce bacterial bioburden in wounds and decrease reliance on systemic antibiotics. aBL induces the generation of reactive oxygen species (ROS) through photoexcitation of endogenous chromophores, causing bacterial damage and death. This study explores the combination of tetracyclines (TCs) with aBL for the treatment of biofilm infections in vitro. Tetracyclines (TCs), including second-generation minocycline (MC), doxycycline (DOCT), and third-generation agents omadacycline (OM) and tigecycline (TG), were evaluated for their ability to enhance bactericidal effects and ROS production during aBL treatment of abiotic biofilm. TCs were tested under dark conditions and with varying aBL light parameters against biofilms of methicillin-resistant Staphylococcus aureus (MRSA), Pseudomonas aeruginosa (PA), and Escherichia coli (E. coli). Results showed that TCs alone were ineffective against these biofilm cultures. However, when combined with aBL either before or after TC treatment, significant enhancement of microbicidal activity was observed. When the aBL is added before the TCs, there was equivalent bactericidal effect, indicating that TCs primary action against biofilms were not as photosensitizers. These findings suggest that aBL can significantly enhance the antimicrobial activity of TCs, potentially offering a new effective approach to treating biofilm-associated infections and combating AMR when aBL is applicable. [ABSTRACT FROM AUTHOR]