Copper contamination of natural waters is a global problem that affects ecosystems and public health, yet this metal is an essential micronutrient and has important applications. The efficacy of Staphylococcus epidermidis CECT 4183 as a Cu(II) biosorbent in synthetic solutions and its potential ability to synthesize CuO nanoparticles (CuO-NPs) from its cellular extract was investigated. In addition, the biocidal potential of the nanoparticles was evaluated against five microorganisms. Using response surface methodology, the optimal operating conditions were determined to be biomass dose, 0.2 g/L, and pH 5.5. Equilibrium tests were performed, and biosorption isotherms were obtained for four models with a maximum biosorption capacity of 48.14 mg/g for the Langmuir model. Different spectroscopic and microscopic techniques were used to determine the mechanisms involved in the biosorption process, which was dominated by surface physicochemical interactions with strong involvement of methyl, methylene, carbonyl, amino, and phosphate groups. The techniques also allowed for characterizing the obtained nanoparticles, which had a quasi-spherical morphology and an average size of 14 nm. Finally, biocidal tests showed that the CuO-NPs had a good inhibitory capacity for the microorganisms tested, with minimum inhibitory concentrations (MIC) between 62.5 and 500 µg/mL for bacteria and between 1000 and 2000 µg/mL for yeasts. S. epidermidis CECT 4183 showed good potential for Cu(II) bioremediation and for the synthesis of CuO-NPs with biocidal capacity. S. epidermidis CECT 4183 showed good potential for use in Cu(II) biosorption, and its cell extract presented a high capacity for the green synthesis of CuO-NPs, which at the same time turned out to be good biocidal agents.