Tetracycline and doxycycline are commonly used antibiotics in acne treatment during puberty in humans. The long-term effect of these antibiotics on male reproductive tract development has not been fully elucidated. For this reason we tested the effect of antibiotics on the reproductive parameters of mice males during puberty with the therapeutic dose used in humans, and with lower and higher doses. The outbred mouse strain CD1 with higher heterozygosity was exposed for 14 days at puberty. Adult males at the age of 70 days were used for the measurements. We observed a significant decrease in anogenital distance and thickness of the seminiferous epithelium in the treated animals. Pathological changes in the testes had an impact on sperm quality; a higher number of sperm positively stained with Annexin V and TUNEL and a lower number of acrosome-intact sperm was detected. In conclusion, the treatment of male mice with antibiotics in puberty led to long-lasting effects on reproductive organs and spermatozoa in adult males.