This study compared the efficacy of the egg yolk (EY) and soy lecithin (SL) semen extender during 72-h storage at 4 °C in the presence/absence of streptomycin, penicillin, lincomycin, and spectinomycin. Bull semen samples (n = 12) were obtained and stored in either ANDROMED® (SL extender) or TRILADYL CSS® (EY extender). After 24, 48, and 72 h of storage at 4 °C, bacterial colonies (CFU – colony forming units) were grown and counted on tryptic soy and blood agar, and the bacterial isolates were identified using the MALDI-TOF MS Biotyper. Sperm motility, mitochondrial membrane potential (MMP), membrane and acrosome integrity, sperm DNA fragmentation, and reactive oxygen species (ROS) production were analysed. The results showed that the type of semen diluent could be crucial for the efficacy of used antibiotics, since significantly decreased bacterial occurrence following 48 h or no bacterial growth after 72 h was recorded in the SL-based groups. Even the SL medium itself did not favour bacterial growth, as significantly decreased bacterial load was observed after 48 h (P < 0.01) and 72 h (P <0.000 1). In contrast, the bacterial load in the EY medium without antibiotics significantly increased (P < 0.05). In contrast, an improved ability to preserve sperm quality parameters was observed in EY-extended semen. In particular, sperm motility was significantly higher (P < 0.000 1 and P < 0.001) after 24 h in each EY-based group in comparison to the control (Ctrl), whereas among the SL-based groups, only the spectinomycin-supplemented group presented with a significant motility improvement (P < 0.01) when compared to the Ctrl. Although the EY semen extender provided enhanced sperm quality preservation during the 72-h storage, in the future, the SL medium composition should be improved to match the sperm preservation ability of the EY medium while maintaining its microbial safety.