The aim of this study was to determine the effect of semen extender supplementation with 25 or 50 μM of zinc nanoparticles (ZnNPs) or manganese nanoparticles (MnNPs) on turkey spermatozoa preserved in a liquid state. Twenty turkey ejaculates were obtained from twenty healthy males. The collected semen was preserved at 4 °C for 48 h with or without NPs. Selected qualitative and quantitative parameters of sperm (motility, plasma membrane activity, mitochondrial membrane potential (MMP) and the percentage of sperm demonstrating NO and SOD activity) were examined after 2, 24 and 48 h of storage. Sperm motility and MMP decreased in semen preserved with ZnNPs at each time point of the analysis. However, all spermatozoa remained viable throughout storage. In contrast, membrane integrity and mitochondria activity (p ≤ 0.05) increased, and the highest SOD activity (p ≤ 0.05) was observed in semen preserved with MnNPs. The addition of MnNPs to the semen extender could potentially improve the parameters of turkey semen during prolonged storage.