This study describes a comparative in vitro study of the toxicity behavior of zinc oxide (ZnO) nanoparticles and micro-sized particles. The study aimed to understand the impact of particle size on ZnO toxicity by characterizing the particles in different media, including cell culture media, human plasma, and protein solutions (bovine serum albumin and fibrinogen). The particles and their interactions with proteins were characterized in the study using a variety of methods, including atomic force microscopy (AFM), transmission electron microscopy (TEM), and dynamic light scattering (DLS). Hemolytic activity, coagulation time, and cell viability assays were used to assess ZnO toxicity. The results highlight the complex interactions between ZnO NPs and biological systems, including their aggregation behavior, hemolytic activity, protein corona formation, coagulation effects, and cytotoxicity. Additionally, the study indicates that ZnO nanoparticles are not more toxic than micro-sized particles, and the 50 nm particle results were, in general, the least toxic. Furthermore, the study found that, at low concentrations, no acute toxicity was observed. Overall, this study provides important insights into the toxicity behavior of ZnO particles and highlights that no direct relationship between nanometer size and toxicity can be directly attributed.