Despite multiple research approaches to prevent bacterial colonization on surfaces, device-associated infections are currently responsible for about 50% of nosocomial infections in Europe and significantly increase health care costs, which demands development of advanced antibacterial surface coatings. Here, novel antimicrobial composite materials incorporating zinc oxide nanoparticles (ZnO NP) into biocompatible poly(N-isopropylacrylamide) (PNIPAAm) hydrogel layers are prepared by mixing the PNIPAAm prepolymer with ZnO NP, followed by spin-coating and photocrosslinking. Scanning electron microscopy (SEM) characterization of the composite film morphology reveals a homogeneous distribution of the ZnO NP throughout the film for every applied NP/polymer ratio. The optical properties of the embedded NP are not affected by the matrix as confirmed by UV-vis spectroscopy. The nanocomposite films exhibit bactericidal behavior towards Escherichia coli (E. coli) for a ZnO concentration as low as ≈0.74 μg cm−2 (1.33 mmol cm−3), which is determined by inductively coupled plasma optical emission spectrometry. In contrast, the coatings are found to be non-cytotoxic towards a mammalian cell line (NIH/3T3) at bactericidal loadings of ZnO over an extended period of seven days. The differential toxicity of the ZnO/hydrogel nanocomposite thin films between bacterial and cellular species qualifies them as promising candidates for novel biomedical device coatings.