Zinc oxide nanoparticles (ZnO NPs) are one of the most widely used nanoparticulate materials due to their antimicrobial properties. However, the current use of ZnO NPs is hindered by their potential cytotoxicity concerns, which are likely attributed to the generation of reactive oxygen species (ROS) and the dissolution of particles to ionic zinc. To reduce the cytotoxicity of ZnO NPs, transitional metals are introduced into ZnO lattices to modulate the ROS production and NP dissolution. However, the influence of the doping element, doping concentration, and particle size on the cytotoxicity and antimicrobial properties remains unexplored. This study presents a comprehensive investigation of a library of doped ZnO NPs to elucidate the relationship between their physicochemical properties, antimicrobial activity against Escherichia coli (E. coli), and cytotoxicity to mammalian cells. The library comprises 30 variants, incorporating three different dopant metals—iron, manganese, and cobalt—at concentrations of 0.25%, 1%, and 2%, and calcined at three temperatures (350 °C, 500 °C, and 600 °C), resulting in varied particle sizes. These ZnO NPs were prepared by low temperature co-precipitation followed by high-temperature calcination. Our results reveal that the choice of dopant elements significantly influences both antimicrobial efficacy and cytotoxicity, while dopant concentration and particle size have comparatively minor effects. High-throughput UV–visible spectroscopic analysis identified Mn- and Co-doped ZnO NPs as highly effective against E. coli under standard conditions. Compared with undoped ZnO particles, Mn- and Co-doping significantly increased the oxidative stress, and the Zn ion release from NPs was increased by Mn doping and reduced by Fe doping. The combined effects of these factors increased the cytotoxicity of Mn-doped ZnO particles. As a result, Co-doped ZnO particles, especially those with 2 wt.% doping, exhibited the most favourable balance between enhanced antibacterial activity and minimized cytotoxicity, making them promising candidates for antimicrobial applications.