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The synthesis of multifunctional photothermal nanoagents for antibiotic loading and release remains a challenging task in nanomedicine. Herein, we investigated a simple, low-cost strategy for the preparation of CuS-BSA nanoparticles (NPs) loaded with a natural enzyme, lysozyme, as an antibacterial drug model under physiological conditions. The successful development of CuS-BSA NPs was confirmed by various characterization tools such as transmission electron microscopy (TEM), X-ray diffraction (XRD), Raman spectroscopy, and X-ray photoelectron spectroscopy (XPS). Lysozyme loading onto CuS-BSA NPs was evaluated by UV/vis absorption spectroscopy, Fourier-transform infrared spectroscopy (FTIR), zeta potential, and dynamic light scattering measurements. The CuS-BSA/lysozyme nanocomposite was investigated as an effective means for bacterial elimination of B. subtilis (Gram-positive) and E. coli (Gram-negative), owing to the combined photothermal heating performance of CuS-BSA and lysozyme release under 980 nm (0.7 W cm−2) illumination, which enhances the antibiotic action of the enzyme. Besides the photothermal properties, CuS-BSA/lysozyme nanocomposite possesses photodynamic activity induced by NIR illumination, which further improves its bacterial killing efficiency. The biocompatibility of CuS-BSA and CuS-BSA/Lysozyme was elicited in vitro on HeLa and U-87 MG cancer cell lines, and immortalized human hepatocyte (IHH) cell line. Considering these advantages, CuS-BSA NPs can be used as a suitable drug carrier and hold promise to overcome the limitations of traditional antibiotic therapy.