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Poly(methyl methacrylate) (PMMA) resins are widely used in medical and dental applications. Their susceptibility to bacterial biofilm formation poses significant challenges related to material degradation and infection risk. This study investigated the effects of Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) biofilms on PMMA resin surface properties over a 45‐day period at 35°C. The study examined various parameters including biofilm adhesion, morphology, surface roughness, hydrophobicity, solid fraction, and zeta potential. PMMA resin specimens were inoculated with bacteria and incubated for 45 days. Biofilm adhesion was visually assessed, while surface characterization was conducted using scanning electron microscopy (SEM), atomic force microscopy (AFM), roughness analysis, contact angle measurements, solid fraction determination, and zeta potential analysis. The P. aeruginosa and S. aureus isolates were selected based on their biofilm‐positive characteristics, which were further confirmed using Congo red and biofilm formation assays through crystal violet staining and spectrophotometric analysis. The results demonstrated robust biofilm adhesion on PMMA surfaces. SEM and AFM imaging revealed textured surfaces with elevated structures and depressions within the biofilm matrix. Biofilm‐exposed resins exhibited significantly increased roughness (Ra = 164.5 nm, Rq = 169.5 nm) and hydrophobicity (mean angle = 85.5°−90.5°) compared to control samples (Ra = 38–50 nm, angle = 55°). Solid fraction measurements indicated a denser biofilm matrix on exposed resins (0.908) compared to controls (0.65). Additionally, zeta potential values were more negative for biofilm‐exposed resins (mean = −84.2 mV) than controls (−45.0 mV). These findings underscore the substantial alterations in PMMA resin surface properties induced by bacterial biofilms, emphasizing the critical need for strategies to prevent biofilm formation and mitigate associated risks in healthcare settings. Future research should focus on developing anti‐biofilm coatings or treatments to preserve the integrity and functionality of PMMA materials. [ABSTRACT FROM AUTHOR]