The current state of multidrug resistance among Mycobacterium bovis (M.bovis) isolates originating from cattle in Egypt underscores the need for control measures based on accurate diagnostic approaches. Conducting antibiogram surveillance of M.bovis using advanced molecular techniques can greatly contribute to the prevention of transmission of multidrug-resistant (MDR) strains to humans. In this study, a total of 153 tuberculous lesion samples were collected from  slaughtered cows in the abattoir  and subjected to bacteriological examination. Acid-fast bacilli were detected in 54.2% of samples using ZN staining, while M.bovis was isolated from 65.4% of samples through culture. Out of the 65 M.bovis isolates confirmed by PCR, 25 showed resistance to at least one anti-tuberculous drug. The resistance rates to EMB, STR, INH, and RIF were 3.1%, 20%, 30.8%, and 32.3% respectively. MDR was detected in 21.5% of isolates. DNA sequencing of six rifampicin-resistant isolates revealed three specific mutations (H526Y, S531L, and D516V). These findings highlight the importance of investigating ante-mortem M.bovis infection in cattle to minimize public health risks associated with bovine tuberculosis.