Two ZnO materials of spherical hierarchical morphologies, with hollow (ZnOHS) and solid cores (ZnOSS), were obtained through the hydrolysis of zinc acetylacetonate in 1,4-butanediol. The nature of the defects and surface reactivity for the two ZnO materials were investigated through photoluminescence, X-ray photoelectron spectroscopy, and electron paramagnetic resonance (EPR) spectroscopy proving the coexistence of shallow and deep defects and, also, the presence of polyol byproducts adsorbed on the outer layers of the ZnO samples. The EPR spectroscopy coupled with the spin-trapping technique showed that the surface of the ZnO samples generates reactive oxygen species (ROS) like hydroxyl (•OH) and singlet oxygen (1O2) as well as carbon-centered radicals. The ZnO materials exhibited a wide spectrum of antimicrobial activity, being active against Gram-positive, Gram-negative, and fungi strains, both in planktonic and, more importantly, adherent growth states. The decrease of antimicrobial efficiency in the presence of a ROS scavenger (mannitol) and the decrease of the cell viability with the ROS level suggest that one of the mechanisms that governs both the antimicrobial and cytotoxic activities on human liver cells is ROS-mediated. However, at active antimicrobial concentrations, the biocompatibility of the tested materials is very good.