Polymethyl methacrylate (PMMA)/Palm oil/ZnO nanocomposite films of concentrations 0, 0.5, 1, 2, 5, 10, and 15 wt% were prepared by solution casting technique. Tween 80 was used as a surfactant. The prepared films were characterised by different techniques in addition to antimicrobial and cytotoxicity tests. The scanning electron microscope (SEM) micrographs of fractured surfaces of the films showed that palm oil (PO) and Tween 80 enhanced ZnO NPs dispersion. An excess ZnO loading led to polymer saturation with ZnO NPs which accumulate on PMMA surface. X-ray diffraction (XRD) measurements confirmed SEM results as the crystallinity increased by ZnO NPs loading and decreased by excess loading. In addition, the presence of PO and Tween 80 enhanced thermal stability of pure PMMA and the optimum concentration is 0.5 wt% ZnO NPs. Further, the permittivity (ε′), dielectric loss (ε′′), and electrical conductivity (σ) were investigated. It was found that PO addition increased the values of ε′, ε′′, and σ of PMMA. Upon ZnO loading, these values increased up to 10 wt% ZnO NPs then decreased upon reaching 15 wt% ZnO NPs. The conductivity values revealed that the prepared nanocomposites can perform as antistatic materials for lower ZnO content and electrostatic dissipation application at 10 wt% ZnO. The antimicrobial and cytotoxicity studies revealed that the prepared films are nontoxic and the antimicrobial properties of the films against Staphylococcus aureus, Enterococcus faecalis and Candida albicans were enhanced by PO and ZnO NPs addition.