Antimicrobial resistance is a threat to public health that requires our immediate attention. With increasing numbers of microbes that are becoming resistant to routinely used antimicrobials, it is vital that we look to other, non-traditional therapies for the treatment of infections. Antimicrobial blue light (aBL) is an innovative approach that has demonstrated efficacy for the inactivation of an array of microbial pathogens. In the present study, we investigated the potential for resistance development to aBL in Gram-negative pathogenic bacteria by carrying out multiple aBL exposures on bacteria. In the first aBL exposure, clinical isolates of Pseudomonas aeruginosa, Acinetobacter baumannii, and uropathogenic Escherichia coli [10⁷ colony forming units/mL (CFU/mL)] were irradiated in phosphate-buffered saline with aBL at 405 nm until a >99.99% reduction in bacterial viability was achieved. Irradiation was then repeated for each bacterial species over 20 cycles of aBL exposure. The potential for resistance development to aBL was also investigated in vivo, in superficial mouse wounds infected with a bioluminescent strain of P. aeruginosa (PAO1; 10⁸ CFU) and irradiated with a sub-curative radiant exposures of 108 or 216 J/cm² aBL over 5 cycles of treatment (over 5 days) prior to bacterial isolation from the animal tissue. PAO1 isolated from infected tissue were treated with aBL at 216 J/cm², in vitro, in parallel with unexposed PAO1 or PAO1 isolates from mouse wound infections not treated with aBL. No statistically significant correlation was found between the aBL-susceptibility of bacteria in vitro and the number of cycles of aBL exposure any bacterial species (P ≥ 0.26). In addition, serial exposure of infected mouse wounds to aBL did not result in any change in the susceptibility to aBL of PAO1 (P = 0.97). In conclusion, it is unlikely that sequential exposure to aBL will result in aBL-resistance in Gram-negative bacteria. Also, multiple aBL treatments may potentially be administered to an infected wound without resistance development becoming a concern.