Bacteriospermia has become a serious factor affecting sperm quality in swine breeding, this is why antibiotics (ATBs) are a critical component of semen extenders. Due to ever-increasing antimicrobial resistance, the aim of this study was to assess the efficiency of selected commercially available semen extenders to prevent a possible bacterial contamination of boar ejaculates. Three Androstar Plus extenders containing different combinations of antibiotics were used to process ejaculates from 30 healthy Duroc breeding boars. Androstar Plus without antibiotics was used as a control. The extended samples were stored at 17 °C for 72 h. Sperm motility, viability, mitochondrial activity, DNA integrity and oxidative profile of each extended sample were assessed following 24 h, 48 h and 72 h. Furthermore, selective media were used to quantify the bacterial load and specific bacterial species were identified with matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The results indicate that semen extenders enriched with ATBs ensured a significantly higher preservation of the sperm quality in comparison to the ATB-free control. The total bacterial count was significantly decreased in the extenders supplemented with ATBs (p < 0.001), however gentamycin alone was not effective enough against Gram-positive bacteria, while a few colonies of Enterococcus hirae, Bacillus subtilis and Corynebacterium spp. were present in the samples extended in the presence of a triple combination of ATBs. In conclusion, we may suggest that semen extenders enriched in antibiotics were not able to fully eliminate the bacteria present in the studied samples. Furthermore, selection of suitable antibiotics for semen extension should be accompanied by adequate hygiene standards during the collection and handling of boar ejaculates.