are among the most frequent bacteria known to cause biofilm-related infections. Pathogenic biofilms represent a global healthcare challenge due to their high tolerance to antimicrobials. In this study, water soluble polyethylene glycol (PEG)-coated gold nanospheres (28 ppm) and nanostars (15 ppm) with electrostatically adsorbed photosensitizer (PS) Toluidine Blue O (TBO) ∼4 μM were successfully synthesized and characterized as PEG-GNPs@TBO and PEG-GNSs@TBO. Both nanoconjugates and the TBO 4 μM solution showed remarkable, if similar, antimicrobial photodynamic inactivation (aPDI) effects at 638 nm, inhibiting the formation of biofilms by two strains: a clinical methicillin-resistant (MRSA) isolate and () RP62A. Alternatively in biofilm eradication treatments, the aPDI effects of PEG-GNSs@TBO were more effective and yielded a 75% and 50% reduction in viable count of MRSA and RP62A preformed biofilms, respectively and when compared with untreated samples. This reduction in viable count was even greater than that obtained through aPDI treatment using a 40 μM TBO solution. Confocal laser microscopy (CLSM) and scanning electron microscope (SEM) images of PEG-GNSs@TBO's aPDI treatments revealed significant changes in the integrity and morphology of biofilms, with fewer colony masses. The generation of reactive oxygen species (ROS) upon PEG-GNSs@TBO's aPDI treatment was detected by CLSM using a specific ROS fluorescent probe, demonstrating bright fluorescence red spots across the surfaces of the treated biofilms. Our findings shine a light on the potential synergism between gold nanoparticles (AuNPs) and photosensitizers in developing novel nanoplatforms to target biofilm related infections.