Conventional antibacterial surfaces are becoming less effective due to the emergence of multidrug resistant bacteria; in addition, difficulties related to the accumulation of killed bacteria are generally encountered. To circumvent these problems, in the present work, an antibiotic-free and regenerable antibacterial hybrid film with both photothermal bactericidal activity and bacteria-releasing properties was fabricated on diverse substrates by sequential deposition of a gold nanoparticle layer (GNPL) and a phase-transitioned lysozyme film (PTLF). Due to the photothermal effect of the GNPL, the hybrid film was able to kill >99% of attached bacteria under near-infrared laser irradiation in 5 min. Moreover, the topmost PTLF layer could be degraded and detached from the surface by immersion in vitamin C solution for a short period, leading to removal of the killed bacteria and surface regeneration. The surface could be used and regenerated in this way through multiple cycles for long-term effective performance. The surface fabrication process is simple and environmentally friendly, and can be applied to diverse materials. These hybrid films thus offer a viable alternative for the killing and removal of adherent bacteria (particularly multidrug resistant bacteria) on the surfaces of medical devices for in vitro applications.