Incorporating zinc oxide nanoparticles (ZnO NPs) into cement mortars may provide additional functions, e.g., self-cleaning and antibacterial or electroconductive ability. However, these NPs are also known for their potential toxicity. During the life cycle of a cement mortar, various abrasive forces cause the release of admixtures to the natural environment. The effect of the released NPs on model microorganisms has not been extensively studied. Previous studies have shown that nanomaterials may affect various microorganisms’ physiological responses, including changes in metabolic activity, biofilming, or growth rate. In this study, we have focused on evaluating the response of model microorganisms, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans, towards ZnO nanoparticles released from cement mortars in different deterioration scenarios. The addition of ZnO nanoparticles to cement mortars had a noticeable effect on impeding the strength development. We have also detected that depending on the deterioration scenario, the release of ZnO nanoparticles was varied. Our studies have also shown that even though the release of nanoform ZnO could be limited by poor dispersion or the used filtration technique, the eluates have caused slight but statistically significant changes in the physiological features of studied microorganisms showing relatively low toxicity.