Bovine tuberculosis, a disease caused by Mycobacterium bovis (M. bovis), represents a threat challenging animal production as it causes severe economic losses for the livestock sector. In addition, M. bovis infects humans through different routes: respiratory, oral, mucous membranes and injured skin causing human tuberculosis of animal origin. In Egypt, bovine tuberculosis considered as one of the most serious threats affecting cattle and other livestock. In this study, the prevalence of bovine tuberculosis in three Egyptian governorates (one in Upper Egypt and the others in Lower Egypt) was investigated. This was to figure out the status of the disease incidence and its negative impact on animal production and public health. Of 220 tuberculous-suspected cattle, 25 (11.36%) were positive by tuberculin test, (10 from Qena 11.1%, 7 from Elbeheira 11.7% and 8 from Elgharbia 11.4%). By conventional culture techniques, out of 25 TB positive cases, 16 were culture-positive (65.4%). Concerning serology, 28 of 220 serum samples of animals tested by tuberculin test, were positive with ELISA (12.7%). Lymph nodes and tissue samples (N. = 52) were collected from 25 tuberculin positive animals. Of the collected samples, 41 (78.8%) were positive by RT-PCR. Meanwhile, 35 (94.6%) of 37 tested isolates were identified as Mycobacterium species using Mycobacterium tuberculosis complex-specific primers. Our results threw a spot of light on the importance of prompt surveillance and improvement of the existing control strategies and even developing better programs to prevent the dissemination of M. bovis infection among animals and to protect humans, consequently.