Slaughtering should only take place in slaughterhouses supervised by veterinarians and with strict adherence to hygiene protocols to guarantee the production of high-quality meat. This study aimed to compare the hygiene of several slaughterhouses using total aerobic plate count (TAPC) and isolation of specific hygiene-indicating bacteria, such as Staphylococcus spp., Escherichia coli (E. coli), and Salmonella spp. 480 samples comprise swabs collected from various sources such as knives (slaughter and skinning knifes), slaughterhouse premises (wall, floor, and tap water samples), swabs from carcasses , workers' shoes and hands. Based on our findings, the hygiene indicator microorganisms are negatively correlated with the biosecurity level. The TAPC was mainly high in samples collected from slaughterhouse B that had the lowest biosecurity score. The highest frequency of Staphylococcus spp., E. coli, and Salmonella spp. was (94.17%), (54.17%), and (5%), in slaughterhouses B, A, and C respectively. PCR targeting the nuc1 gene was used for molecular confirmation of randomly selected Staphylococcus spp. as S. aureus. Only 60% of S. aureus isolates were positive for the biofilm-forming gene (BAP gene) in PCR. According to serological identification of E. coli isolates, the most popular E. coli serotypes in the cattle slaughterhouses were O44: K74, O142: K86, O119: K69, O164: K– and O26: K60, while S. enterica sub SP Salamae was the most frequent salmonella serotype. In conclusion, good personal hygiene and biosecurity measures serve as the primary safeguard against zoonotic infections in employees and bacterial colonization of livestock.