Whether there is a relationship between quality, DNA methylation, and mitochondrial DNA (mtDNA) copy number in human-derived sperm specimens is unknown. A cohort (n = 118) of male partners of couples who were undergoing fertility assessment because of an idiopathic inability to conceive were recruited. Sperm motility parameters were determined by computer-aided sperm analysis (CASA), while sperm quality was assessed using World Health Organization criteria, mtDNA copy number was measured by real-time PCR, and DNA methylation patterns were analyzed employing high-melting resolution PCR and bisulfite sequencing PCR. The mtDNA copy number negatively correlated with semen parameters, including sperm motility, concentration, morphology, progression, and motion characteristics (r for −0.19 to −0.54; P < 0.05 for all). As a surrogate marker for global DNA methylation, LINE-1 negatively correlated with sperm motility (r = −0.25; P = 0.009). Meanwhile, after adjustment for age, length of abstinence, smoking, and alcohol intake, there was a suggested association for increased LINE-1 methylation and mtDNA copy number tertiles versus sperm motility (odd ratios were 1.0, 2.6, and 4.7, and 1.0, 2.5, and 4.9, respectively). Altered mtDNA copy number and DNA methylation may serve as genetic and epigenetic markers to assess human sperm quality together with CASA parameters.