The main objective of this investigation was to study the implications of incorporating zinc oxide nanoparticles into the matrix of a bioactive glass for the bioactivity and structural properties of the deposited coating. ZnO-containing bioactive glass was coated on an AISI 316L stainless steel substrate using the electrophoretic deposition technique. AISI 316L stainless steel is a biomedical grade steel, which is widely used in different biomedical applications. For the electrophoretic deposition, voltages and times were chosen in the range of 15–40 V and 15–120 min, respectively. The microstructure, phase composition, and surface roughness of coated samples were analyzed in this investigation. Moreover, the corrosion behavior and the MTT (mitochondrial activity) of samples were studied. Results showed a uniform distribution of elements such as silicon and calcium, characteristic of bioactive glass 58S5, in the coating as well as the uniform distribution of Zn inside the ZnO-containing samples. The findings showed that the deposited ZnO-containing bioactive glass is a hydrophilic surface with a relatively rough surface texture. The results of the MTT and antibacterial effects showed that the deposited layers have promising cell viability.