Cobalt nanoparticles (CoNPs) released from metal-on-metal implants have caused considerable concern. Oxidative stress is associated with the mechanism underlying cobalt-induced cytotoxicity and genotoxicity. The indolamine melatonin exhibits protective effects against damage induced by metals. The present study investigated the in vitro effects of melatonin on the cytotoxicity and genotoxicity induced by CoNPs. CoNPs (20–50 nm in diameter) were employed in the present study. NRK rat kidney cells were exposed to various concentrations of CoNPs for different durations. The results of the current study demonstrated that CoNPs significantly increased reactive oxygen species (ROS) production and reduced cell viability, as determined by dichlorofluorescein diacetate, and Cell Counting Kit-8 and lactate dehydrogenase leakage assays, respectively. Furthermore, western blot analysis demonstrated that CoNPs led to an increase in the ratio of Bcl-2-associated X/Bcl-2, and the expression of cleaved caspase-3 was upregulated, which indicated increased apoptosis levels. Genotoxicity was detected by a comet assay, which revealed a significant induction in DNA damage, as determined by increases in the tail DNA % and olive tail moment. Phosphorylated-histone H2AX foci analyses by immunofluorescence also demonstrated that CoNPs induced DNA-double strand breaks. However, cellular treatment with melatonin reduced the effects of CoNPs on NRK cells by reducing the production of ROS. The results of the present study demonstrated that CoNPs induced cytotoxicity and genotoxicity by increasing oxidative stress, and melatonin may have pharmacological potential in protecting against the damaging effects of CoNPs following total hip arthroplasty.