Catheter-associated urinary tract infection (CAUTI) is the most common healthcare-associated infection. The increasing rates of antibiotic resistance in CAUTI need the development of alternative therapeutic approaches. Gold nanoparticles (AuNPs) are particularly appealing due to their inherent inertness and low cytotoxicity, making them ideal for a wide range of biological and antibacterial applications. Sericin protein, derived from silkworm cocoons (Bombyx mori), is regarded as a byproduct but has found value in biomedical applications due to its processability, biocompatibility, and biological inertness. However, the molecular mechanisms underlying the antibacterial properties of sericin-conjugated AuNPs (AuNP-sericin) in human urine remain uncharacterized, and guidance for their appropriate application in catheters is limited. Forty catheter-associated urine samples were collected for urinalysis and bound to AuNP-sericin for a molecular mechanism study using proteomics. AuNP-sericin accumulation on microorganism cell surfaces via electrostatic interactions, as evidenced by transmission electron microscope. This situation may disrupt membranes, block nutrient access, and thus interfere with microbial metabolic pathways. Proteomics investigation confirmed that AuNP-sericin can restrict microbial growth by trapping their membranes, interfering with translation and metabolism. These qualities may inhibit colonization and biofilm development, lowering antibiotic use and CAUTI rates, which are a leading cause of hospitalization.