The role of antibiotics (AB) in semen extenders as a potential contribution to the global antimicrobial resistance threat is emerging. Here, we establish an AB-free hypothermic preservation strategy for boar semen and investigate its impact on sperm function, microbial load and fertility after artificial insemination (AI). Spermatozoa (12 boars) preserved in AB-free AndroStar Premium extender at 5 °C maintained high motility, membrane integrity, and a low DNA-fragmentation index throughout 72 h storage and results did not significantly differ from controls stored at 17 °C in extender containing AB (p = 0.072). Likewise, kinetic response of spermatoza to the capacitation stimulus bicarbonate during 180 min incubation in Tyrode's medium did not differ from 17 °C-controls. In a competitive sperm oviduct binding assay, binding indices did not differ between semen stored for 72 h AB-free at 5 °C and 17 °C-controls (n = 6 boars). Bacterial load < 103 CFU/ml after 72 h was measured in 88.9% of samples stored at 5 °C AB-free compared to 97.2% in 17 °C-controls (n = 36 semen pools, 23 boars). Fertility traits of 817 females did not differ significantly between the two semen groups (p > 0.05). In conclusion, a hypothermic semen preservation strategy is presented which offers antibiotic-free storage of boar semen doses.