The use of polymer-based medical devices has reduced material costs, improved quality of care, and increased device biocompatibility. However, polymer devices are susceptible to fouling by foreign bacteria, leading to fatal hospital-acquired infections. Antibiotic-resistant bacterial strains have created the need to focus on decreasing initial adhesion of bacteria to the polymer surface. Previous research has shown that both material stiffness and chemistry impact adhesion of structurally different bacteria to polymeric surfaces. Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus demonstrated greater adhesion on stiffer, hydrophilic poly(ethylene glycol) dimethacrylate (PEGDMA). These findings established a core trifecta: mechanical properties, material chemistry and biological components impact bacterial adhesion to biomaterials. I will report how the attachment of E. coli and S. aureus are impacted by the material stiffness of hydrophobic polydimethylsiloxane (PDMS) gels. More bacteria adhered to softer PDMS gels, which was opposite of the observed trend on PEGDMA hydrogels. Next, by spin-coating thin PDMS gels, I investigated if their stiffness at different thicknesses (10 µm, 35 µm, 100 µm) impacted adhesion. It was demonstrated that as thickness decreased, bacterial adhesion increased, same as the trend observed on PEGDMA hydrogels. Next, biomaterials were designed to mimic usage in clinical settings, symbolizing PEG as an antifouling coating atop a PDMS catheter. These “layered” gels, featured PEGDMA hydrogels (15-60 µm thick) of different stiffnesses deposited onto spin-coated PDMS gels of various stiffnesses and a consistent thickness (100 µm). The attachment of E. coli and S. aureus were assessed on these layered gels to further elucidate the role of surface chemistry. Finally, a collaboration used genetic analysis and CRISPRi tools to identify and repress important genetic targets involved in E. coli adhesion, and adhesion assays demonstrated that their adhesion phenotypes can be controlled. The results of this dissertation have elucidated important findings regarding how surface mechanics, surface chemistry and biology each are important pillars of understanding how bacteria interact with surfaces, and how E. coli adhesion-associated genes have been identified and repressed with CRISPRi systems. These results hold potential to guide the design of antifouling polymeric biomaterial devices and CRISPRi treatments to prevent hospital acquired infections.