Nosocomial infections, a prevalent issue in intensive care units due to antibiotic overuse, could potentially be addressed by metal oxide nanoparticles (NPs). However, there is still no comprehensive understanding of the impact of NPs’ size on their antibacterial efficacy. Therefore, this study provides a novel investigation into the impact of ZnO NPs’ size on bacterial growth kinetics. NPs were synthesized using a sol–gel process with monoethanolamine (MEA) and water. X-ray diffraction (XRD), transmission electron microscopy (TEM), and Raman spectroscopy confirmed their crystallization and size variations. ZnO NPs of 22, 35, and 66 nm were tested against the most common nosocomial bacteria: Escherichia coli, Pseudomonas aeruginosa (Gram-negative), and Staphylococcus aureus (Gram-positive). Evaluation of minimum inhibitory and bactericidal concentrations (MIC and MBC) revealed superior antibacterial activity in small NPs. Bacterial growth kinetics were monitored using optical absorbance, showing a reduced specific growth rate, a prolonged latency period, and an increased inhibition percentage with small NPs, indicating a slowdown in bacterial growth. Pseudomonas aeruginosa showed the lowest sensitivity to ZnO NPs, attributed to its resistance to environmental stress. Moreover, the antibacterial efficacy of paint containing 1 wt% of 22 nm ZnO NPs was evaluated, and showed activity against E. coli and S. aureus.