Tuberculosis (TB) has been an important public health concern in Bangladesh. The most common cause of human TB is Mycobacterium tuberculosis, while bovine TB is caused by Mycobacterium bovis. The objective of this study was to determine the frequency of TB in individuals with occupational exposure to cattle and to detect Mycobacterium bovis among cattle in slaughterhouses in Bangladesh. Between August 2014 and September 2015, an observational study was conducted in two government chest disease hospitals, one cattle market, and two slaughterhouses. [Correction added on 27 June 2023, after first online publication: In the preceding sentence, the year "2014" has been added after the word "August".] Sputum samples were collected from individuals who met the criteria for suspected TB and had been exposed to cattle. Tissue samples were collected from cattle that had low body condition score(s). Both humans and cattle samples were screened for acid-fast bacilli (AFB) by Ziehl-Neelsen (Z-N) staining and cultured for Mycobacterium tuberculosis complex (MTC). Region of difference (RD) 9-based polymerase chain reaction (PCR) was also performed to identify Mycobacterium spp. We also conducted Spoligotyping to identify the specific strain of Mycobacterium spp. Sputum was collected from a total of 412 humans. The median age of human participants was 35 (IQR: 25-50) years. Twenty-five (6%) human sputum specimens were positive for AFB, and 44 (11%) were positive for MTC by subsequent culture. All (N = 44) culture-positive isolates were confirmed as Mycobacterium tuberculosis by RD9 PCR. Besides, 10% of cattle workers were infected with Mycobacterium tuberculosis in the cattle market. Of all TB (caused by Mycobacterium tuberculosis) infected individuals, 6.8% of individuals were resistant to one or two anti-TB drugs. The majority of the sampled cattle (67%) were indigenous breeds. No Mycobacterium bovis was detected in cattle. We did not detect any TB cases caused by Mycobacterium bovis in humans during the study. However, we detected TB cases caused by Mycobacterium tuberculosis in all humans, including cattle market workers.