The association of leukocytospermia with male fertility is still under debate. Our objective is to evaluate the impact of leukocytospermia on nuclear and mitochondrial DNA (mtDNA), on semen parameters as well as on biomarkers, which include oxidative stress and inflammatory cytokines, in Tunisian infertile men. The studied patients were divided into two groups: patients without leukocytospermia (Group 1) and patients with leukocytospermia (Group 2). DNA fragmentation evolved significantly in group 2 (31.41%) compared to group 1 (14.68%) ; (p < 0.001). A total of 115 nucleotide substitutions in mitochondrial DNA were depicted, among which 113 were previously identified. The number of substitutions was more elevated in group 2. Leukocytospermic group had significantly higher MDA (nmole/ml) levels than patients without leukocytospermia (34 ± 24.43 vs 18.94 ± 15.96 ; p  = 0.001), GSH (µg/ml) levels were also higher compared to the control group (126.53 ± 22.87 vs 79.4 ± 19.38 ; p < 0.001), SOD (U/mg of protein) levels were higher but without reaching the statistical significance (89.74 ± 74.85 vs 67.56 ± 37.11 ; p = 0.25) ; whereas seminal CAT (µmole H₂O₂/min/mg of protein) levels were lower in this group (10.66 ± 14.32 vs 27.35 ± 25.28 ; p = 0.012). No statistically significant differences between the two groups of patients were found in the levels of cytokines. However, IL-8 level was positively correlated with DNA fragmentation and negatively correlated with vitality. These findings confirm the leading role of leukocytospermia in the perturbation of semen parameters and in sperm DNA damage.