Bacterial infections pose a serious problem, often necessitating the use of antimicrobials in substantial quantities. Furthermore, microbial resistance has significantly escalated alongside the rapid adaptation of bacteria to antimicrobial agents. This research aims to explore alternative solutions for diminishing
Staphylococcus aureus
and
Pseudomonas aeruginosa
bacteria through photodynamic therapy. Gold nanoparticles (AuNPs) were synthesized using a laser ablation technique and applied as photosensitizers at concentrations of 10.0, 15.0, and 21.5 μg/mL. A 405 nm blue diode laser was employed for photodynamic inactivation (PDI) against
Staphylococcus aureus
and
Pseudomonas aeruginosa
. Antibacterial efficacy was evaluated by quantifying bacterial reduction through Total Plate Count (TPC) in CFU/mL and confirmed using the disc diffusion assay. The research results indicated that adding 21.5 µg/mL of AuNPs photosensitizer at a volume of 50 µL led to a notable reduction in bacterial colonies. The most effective reduction in
Staphylococcus aureus
colonies was achieved with 150 s of laser exposure at an energy level of 2.87 J/cm
2
, reducing (80.3± 5.39)%. Similarly, the optimal reduction in
Pseudomonas aeruginosa
colonies occurred with 180 s of laser exposure at an energy level of 3.44 J/cm
2
, reducing (78.70±4.50)%. Blue laser irradiation at 405 nm combined with 21.5 μg/mL AuNPs effectively reduced
Staphylococcus aureus
by (80.37 ± 5.39)% in 150 s and
Pseudomonas aeruginosa
by (78.70 ± 4.50)% in 180 s, demonstrating its potential as an alternative antibacterial therapy.