Bovine tuberculosis (BTB) is a zoonotic illness of significant public health concern. This research aims to investigate the prevalence of BTB and its risk factors in Upper Egypt governorates. A total of 600 cattle (200 from each of New Valley, Qena, and Aswan) were tested using a single intradermal cervical tuberculin test (SICTT), and the positive animals were slaughtered at the central abattoirs of each governorate. The tissues of the affected carcasses were inspected by Ziehl-Neelsen (ZN) staining and culturing, and confirmation of results was achieved through ELISA and PCR. The findings revealed that 2.3% of inspected animals tested positive by the SICTT. Qena recorded the highest rate of tuberculin-positive animals at 3.5%. BTB was found to be statistically associated with sex, body condition, age, breed, and yard density, and the thoracic organs and their lymph nodes were mostly affected. ZN staining identified only 85.7% of the isolates as acid-fast bacilli. ELISA results indicated that 78.6% of positive tuberculin animals were also positive for bovine-purified protein derivative antigen and 71.4% were positive for commercial polypeptide antigen. Furthermore, there was no significant correlation between the molecular identification of Mycobacterium bovis using tissue samples or isolates, and the PCR results confirmed the occurrence of Mycobacterium bovis DNA in A8 of each examined category. The findings underscore the crucial role of slaughterhouses in providing essential data for monitoring BTB epidemiology within specific regions. These insights are pivotal for forming strategies and implementing effective regulation and prevention measures.