Toxoplasma gondii is an obligate intracellular protozoan parasite that can infect virtually any nucleated cell within human and other endoderm animal tissue, including male reproductive organs. Herein, we investigate the capacity of T. gondii tachyzoites to infect and proliferate within the testes and epididymis and examine the resulting impact on human spermatozoa structure and functionality. We confirmed that T. gondii tachyzoites colonise and proliferate within the testes and epididymis, altering the tissue structural homeostasis, and causing immune cell infiltration and cellular damage. In addition to demonstrating that T. gondii remains infective within the testes and epididymis, in vitro experiments demonstrated a direct interaction between T. gondii tachyzoites and human spermatozoa. This resulted in a significant proportion of headless spermatozoa. Scanning and transmission electron microscopy revealed structural defects in spermatozoa, such as twisted tails and plasma membrane disruptions. Moreover, T. gondii tachyzoites triggered the loss of mitochondrial membrane potential (MMP) in spermatozoa without modulating reactive oxygen species (ROS) concentrations, and triggered cell death, pointing at mitochondrial dysfunction as a potential mechanism mediating spermatozoan damage. Our findings suggest that T. gondii infection can have profound implications for male fertility by directly damaging spermatozoa and altering testicular and epididymal structures. The study underscores the need for further research to elucidate the long-term impact of T. gondii on male reproductive health, particularly in the context of iatrogenic infertility. Given the widespread seroprevalence of T. gondii in the human population, our research emphasises the importance of considering parasitic infections in diagnosing and managing male infertility in the field of andrology.