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Antimicrobial blue light (aBL) treatment is considered low risk for the development of bacterial resistance and tolerance due to its multitarget mode of action. The aim of the current study was to demonstrate whether tolerance development occurs in Gram-negative bacteria. We evaluated the potential of tolerance/resistance development in <i>Escherichia coli</i>, <i>Klebsiella pneumoniae</i>, and <i>Pseudomonas aeruginosa</i> and demonstrated that representative Gram-negative bacteria may develop tolerance to aBL. The observed adaption was a stable feature. Assays involving <i>E. coli</i> K-12 <i>tolC</i>-, <i>tolA-</i>, <i>umuD-</i>, and <i>recA</i>-deficient mutants revealed some possible mechanisms for aBL tolerance development.