Sperm motility is a key indicator of male fertility. Decreased motility, or asthenozoospermia, highlights the need for understanding male fertility challenges. This study was designed to evaluate the effects of in vitro use of follicle-stimulating hormone (FSH) on various sperm parameters, sperm DNA integrity, and mitochondrial membrane potential. Semen samples were obtained from 20 asthenozoospermic men. Each sample was divided into control and case groups. In the case group, samples were incubated with FSH at 30 mIU/mL for 1 h, while in the control group, the samples were incubated for 1 h without treatment. Sperm parameters, DNA fragmentation, and mitochondrial membrane potential were assessed in the two groups based on WHO 2021 criteria. Progressive motility and rapid progressive motility were higher in the case group compared to the control. However, FSH did not have a significant effect on morphology, viability, DNA fragmentation, or mitochondrial membrane potential in either group. In conclusion, FSH effectively enhanced sperm motility without compromising sperm DNA integrity or mitochondrial membrane potential (MMP). Therefore, FSH can be recommended as a safe and effective option for sperm selection in patients with asthenozoospermia.