The growing prevalence of biofilm-associated multi-drug resistant (MDR) bacteria necessitates the innovation of non-traditional approaches to improve the effectiveness of mainstay antibiotics. Here, we evaluated the use of gold nanoparticle (GNP)-targeted pulsed laser therapy to enhance antibiotic efficacy against in vitro methicillin-resistant Staphylococcus aureus (MRSA) and MDR Pseudomonas aeruginosa biofilms. Treatment with antibody-conjugated GNPs followed by nanosecond-pulsed laser irradiation at 532 nm (~1.0 J/cm²) dispersed 96–99% of the biofilms relative to controls. GNP-targeted laser therapy combined with gentamicin or amikacin caused a synergistic 4- and 5-log reduction in the viability of MRSA and P. aeruginosa biofilms, respectively, whereas GNP-targeted laser therapy or antibiotics alone decreased biofilm viability by only ~1 log. Notably, GNP-targeted laser therapy was able to increase the antibiotic susceptibility of the biofilms to the level of drug sensitivity observed in planktonic MRSA and P. aeruginosa cultures, further indicating effective biofilm dispersal via this novel approach.