Multi-drug antibiotic resistance ofSerratia (S.) marcescensandKlebsiella (K.) oxytocain boar semen is an emerging threat to pig reproduction and the environment. The aim of this study is to examine the efficiency of a novel hypothermic preservation method to inhibit the growth of these bacterial species in extended boar semen and to maintain the sperm quality. The semen samples extended in an antibiotic-free Androstar Premium extender were spiked with ~102CFU/mL ofS. marcescensorK.oxytoca. Storage at 5 °C for 144 h inhibited the growth of both bacterial species and maintained the sperm quality, whereas bacterial counts increased to more than 1010CFU/mL in the 17 °C samples used as positive controls. This was accompanied by an increase in the sperm agglutination and the loss of motility and membrane integrity. We conclude that hypothermic storage is a promising tool to combat resistant bacteria in boar semen and to contribute to the One Health approach.