The uncertainties in ZnO-mediated toxicity and particle stability in a biological system remain a challenge and mitigate against deployment as next-generation nanoparticles (NPs), especially in biomedical applications. With that perspective, the present study investigates the surface chemical properties of ZnO NPs coated with three different surfactant biomolecules, namely polyethylene glycol (PEG), cetyltrimethylammonium bromide (CTAB), and sodium dodecyl sulfate (SDS) to control the toxicity-induced potentials. On the testing of the surface-functionalized ZnO NPs, notable changes in the particle sizes, morphology, zeta potential, and hydrodynamic size compared to the pure ZnO NPs are observed. In addition, FTIR spectroscopy, TGA, XRD, XPS, and HRTEM analysis showed significant changes in the surface structures and surface functional groups of the three different ZnO NPs on surface functionalization. Following the physical characterization, the cell viability of rat liver BRL-3A-treated ZnO–PEG, ZnO–CTAB, and ZnO–SDS compared to pure ZnO NPs ( ZnO–CTAB > ZnO–PEG, i.e., anionic > cationic > non-ionic. Overall from the analysis, the study stresses the importance of having a suitable surface ligand for the ZnO NPs so as to use them in the biomedical sector.