Sperm DNA fragmentation is associated with poor sperm quality and reproductive outcomes. Free radicals are a significant cause of DNA fragmentation, with mitochondria being the primary intrinsic source. To investigate the relationship between midpiece measurements containing mitochondria and sperm DNA fragmentation, and to determine the ideal midpiece area. Demographic data, semen analysis results, and DNA fragmentation values were prospectively collected from 239 men with infertility complaints. Detailed analyses and morphometric measurements were performed on 50 spermatozoa from each patient, totaling 11,950 spermatozoa. The ideal DNA fragmentation index (DFI) cutoff value was calculated. Patients were classified into three subgroups based on midpiece length, midpiece width, and midpiece area measurements. The correlation between mid-piece measurements and DFI was investigated. The cutoff value for DFI was determined as 19.50. The odds ratio evaluating the relationship between morphology status and high DFI was 0.159 (95% CI: 0.089—0.282). It was observed that midpiece length and width have statistically significant but low correlations with DFI, whereas midpiece area shows a higher correlation. Finally, based on DFI values, the ideal midpiece area was between 2.31 and 3.13 µm ² . There is a significant correlation between sperm midpiece area and DFI value, surpassing that of length and width. Future studies may yield important insights by exploring the impact of midpiece area measurements on reproductive outcomes.