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Objective The present study aimed to identify Mollicutes infection in the reproductive system. We also examined the microbiological, biochemical, and antimicrobial profiles of Mollicutes infection, which are potentially associated with clinical reproductive abnormalities causing infertility in couples. Methods Thirty-seven couples who were attending an infertility clinic were enrolled. Detection of genital mycoplasmas was performed in cervicovaginal samples or male urethral swabs. Microbiological culture and biochemical and antimicrobial profiles were characterized using a Mycoplasma kit. The results were associated with reproductive abnormalities, as assessed by medical specialists from the infertility clinic. Results Up to 28.3% of all biological samples (n = 74) showed positive cultures. Bacterial isolates were Ureaplasma urealyticum (71.4%), Mycoplasma hominis (9.5%), or coinfections (19%). Most Mollicutes showed significant resistance to fluoroquinolones, macrolides, and tetracycline; and showed susceptibility to doxycycline, josamycin, and pristinamycin. The presence of resistant strains to any antibiotic was significantly associated with genital abnormalities (χ 2 test, relative risk = 11.38 [95% confidence interval: 5.8–22.9]), particularly in women. The highest statistical association was found for macrolide-resistant strains. Conclusion The microbiological antibiotic resistance profile is epidemiologically associated with abnormalities of the reproductive system in couples attending an infertility clinic.