This study explores how surface and mechanical properties influence biofilm formation in Staphylococcus aureus and Pseudomonas aeruginosa. Foodborne isolates were molecularly identified and assessed for biofilm-forming potential using crystal violet assays, followed by structural analysis through atomic force microscopy (AFM) and scanning electron microscopy (SEM). P. aeruginosa showed faster surface adhesion (τ = 4 s) than cell–cell interactions (τ = 21 s) and exhibited gradual softening from 1.3 MPa to 1.1 MPa over 96 h. Conversely, S. aureus biofilms displayed continuous stiffness reduction (0.9–0.4 MPa) with increasing roughness (Ra = 2.5 nm). Both species demonstrated enhanced surface hydrophobicity correlating inversely with stiffness. SEM revealed dense extracellular matrices and intercellular communication structures. Together, these results highlight species-specific adaptations in biofilm architecture and mechanics, offering new perspectives for designing targeted antibiofilm interventions.