The prevalence of bacterial infections presents a significant challenge in the medical field, demanding effective strategies to impede bacterial adhesion and growth on various surfaces. The conducted study investigates the efficacy of polyelectrolyte multilayers─comprising poly(allylamine hydrochloride) (PAH) and alginate (ALG)─embedded with zinc oxide (ZnO) and copper oxide (CuO) nanoparticles (NPs) to inhibit bacterial adhesion on stainless-steel surfaces. Surface characterization involved zeta potential, contact angle, and roughness assessments. The effect of NP composition, size, and morphology in conjunction with polycation or polyanion terminating multilayers was evaluated against planktonic and surface-adhered Escherichia coli (E. coli) cells. Surfaces with the positively charged PAH-terminating multilayer displayed higher water contact angles (≈ 63°) than the negatively charged ALG-terminating multilayers (≈ 45°). Multilayers containing ZnO NPs showed a significant inhibition of planktonic E. coli growth, >99%. Moreover, complete growth inhibition of surface-adhered E. coli was achieved for multilayers containing both ZnO and CuO. Due to their larger specific surface area, rod-like ZnO NPs displayed higher antibacterial activity. The samples with ALG as the terminating layer showed more substantial antibacterial properties than samples with PAH as the terminating layer. Biocompatibility tests on immortalized human keratinocyte cells revealed good compatibility with multilayers incorporating NPs. In summary, this study underscores the potential of ZnO and CuO NPs within PAH/ALG multilayers for antibacterial applications without compromising their cytocompatibility.