To detect the prevalence of Mycobacterium tuberculosis complex (MTBC) in the lymph nodes of slaughtered cattle collected from selected abattoirs in Eastern Cape Province of South Africa. A total of 376 lymph nodes were collected from slaughtered cattle over a period of 12 months. Certain characteristics (sex, age, body condition score, and breed) were observed to be associated with MTBC among slaughtered cattle. Collected samples were cultured and tested for acid-fast bacilli (AFB). DNA was isolated, purified, and quantified using a spectrophotometer. Quantified DNA was confirmed to be MTBC by multiplex PCR targeting two genes (IS6110 and mpb64). Of the 376 collected lymph nodes, 182 were positive when tested by Ziehl-Neelsen stain and 162 were confirmed positive for MTBC by PCR. MTBC was isolated from lymph nodes with nodular lesions (72.8%, 118/162) and inflamed lymph nodes (27.1%, 44/162). All detected MTBC isolates were positive for region of deletion 1 (RD1). No isolate was detected to have Mycobacterium bovis bacille Calmette-Guérin (BCG). However, 3.1% had M. bovis and 96.9% had M. tuberculosis. The presence of live Mycobacterium strains in slaughtered cattle poses a health risk to beef consumers and abattoir workers.