Although freezing methods have been optimized for preserving sperm integrity, their effectiveness in sustaining bacterial viability is unknown. Therefore, culturing thawed semen samples might not give an accurate picture of the bacteria in the original sample. The aim of this study was to assess how cryopreservation and storage duration influence bacterial populations and the survival of distinct bacterial species. Semen samples were collected from 14 bulls, samples were diluted in equal proportions of antibiotic-free semen extender and transported to the laboratory at 6 °C overnight. Aliquots of semen were cultured within 24 h after semen collection on Plate Count Agar to calculate number of bacteria, and blood agar plates (5 % bovine blood) for identification of bacterial species. The remaining samples were diluted 1:1 in Brain Heart Infusion (BHI) broth with 30 % glycerol and stored at -80 °C. The frozen samples were thawed and cultured for quantification of bacteria as described for fresh semen, after 6 and 13 days at -80 °C. The isolated bacteria were re-cultured on blood agar, incubated for one day at 37 °C before identification by Matrix assisted laser desorption ionization-time of flight mass spectrometry. Total bacterial counts remained consistent across fresh and cryopreserved samples regardless of storage duration. A total of 31 bacterial species were identified, with 20 detected in fresh samples, 16 present after 6 days of storage, and 18 observed after 13 days. Ten species persisted across all time points, while others were unique to a specific sampling day, including nine species on day 1, two species on day 6, and five species on day 13. These findings suggest that while cryopreservation does not alter the overall bacterial load, the survival of individual species varies depending on storage conditions.