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Platelet-rich plasma (PRP) is a pioneering therapy widely used in various medical fields, showing promising outcomes. However, its impact on human sperm quality remains poorly explored among emerging therapies. This study aims to investigate the effect of autologous PRP supplementation on oxidative stress levels and mitochondrial activity in human sperm. PRP was freshly prepared from venous blood and added to each ejaculated semen sample at different concentrations of 2%, 5%, and 10%. Reactive oxygen species (ROS) in spermatozoa were measured after 24 hours of incubation at 37° (5% CO2), using nitro-blue tetrazolium (NBT) test. The MTT test was used to measure the mitochondrial succinate deshydrogenase activity. A total of 180 semen samples were obtained from 15 patients. The supplementation with PRP significantly reduced the reactive oxidative species levels and improved mitochondrial activity in spermatozoa. The level of oxidative stress in sperm was significantly decreased after 24h of incubation with PRP at 2% (p = 0.001), 5% (p = 0.001) and 10% (p = 0.001) when compared to the control group. The succinate dehydrogenase activity was enhanced in the three groups when compared to the control group. It increased from 0.667 ± 0.313 to 0.952 ± 0.499 (p = 0.018), 1.201 ± 0.657 (p = 0.002) and 1.159 ± 0.607 (p = 0.001) after incubation with 2%, 5% and 10% of PRP, respectively. This study has shown that PRP supplementation could be a promising tool to enhance sperm quality against oxidative stress and mitochondrial dysfunction. These findings could be a starting point to investigate the usefulness of PRP in ART procedures.