The goal of the study was to investigate the level of zinc oxide nanoparticles (ZnO NPs) release from polymethyl methacrylate (PMMA)–ZnO nanocomposites (2.5%, 5%, and 7.5% w/w), as well as from the ZnO NPs layer produced on pure PMMA, and the impact of the achieved final ZnO NPs concentration on cytotoxicity, before the potential use as an alternative material for denture bases. The concentration of ZnO nanoparticles released to the aqueous solution of Zn²⁺ ions was assessed using optical emission spectrometry with inductively coupled plasma (ICP-OES). In the control group (pure PMMA), the released mean for ZnO was 0.074 mg/L and for individual nanocomposites at concentrations of 2.5%, 5%, and 7.5% was 2.281 mg/L, 2.143 mg/L, and 3.512 mg/L, respectively. The median for the ZnO NPs layer produced on PMMA was 4.878 mg/L. In addition, in vitro cytotoxicity of ZnO NPs against the human HeLa cell line was determined through the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) dye. The cytotoxicity studies demonstrate that ZnO nanoparticles in the concentrations up to 20 mg/L have no adverse effect on HeLa cells. When compared with the released and cytotoxic concentrations of ZnO NPs, it can be expected that ZnO released from dental prostheses to the oral cavity environment will have no cytotoxic effect on host cells.