Mycobacterium tuberculosis causes the majority of tuberculosis (TB) cases in humans; however, in developing countries, human TB caused by M. bovis may be frequent but undetected. Human TB caused by M. bovis is considered a zoonosis; transmission is mainly through consumption of unpasteurized dairy products, and it is less frequently attributed to animal-to-human or human-to-human contact. We describe the trends of M. bovis isolation from human samples and first-line drug susceptibility during a 15-year period in a referral laboratory located in a tertiary care hospital in Mexico City. Data on mycobacterial isolates from human clinical samples were retrieved from the laboratory’s database for the 2000–2014 period. Susceptibility to first-line drugs: rifampin, isoniazid, streptomycin (STR) and ethambutol was determined. We identified 1,165 isolates, 73.7% were M. tuberculosis and 26.2%, M. bovis. Among pulmonary samples, 16.6% were M. bovis. The proportion of M. bovis isolates significantly increased from 7.8% in 2000 to 28.4% in 2014 (X2trend, p<0.001). Primary STR resistance was higher among M. bovis compared with M. tuberculosis isolates (10.9% vs.3.4%, p<0.001). Secondary multidrug resistance (MDR) rates were 38.5% and 34.4% for M. bovis and M. tuberculosis, respectively (p = 0.637). A rising trend of primary STR monoresistance was observed for both species (3.4% in 2000–2004 vs. 7.6% in 2010–2014; p = 0.02). There is a high prevalence and a rising trend of M. bovis isolates in our region. The proportion of pulmonary M. bovis isolates is higher than in previous reports. Additionally, we report high rates of primary anti-tuberculosis resistance and secondary MDR in both M. tuberculosis and M. bovis. This is one of the largest reports on drug susceptibility of M. bovis from human samples and shows a significant proportion of first-line anti-tuberculosis drug resistance. Human tuberculosis caused by Mycobacterium bovis (HTBMb) is a lesser-known form of the disease. The main route of transmission of HTBMb is the consumption of unpasteurized dairy products, causing mostly extrapulmonary disease. M. bovis is naturally resistant to pyrazinamide, a drug that allows for a shorter treatment course. Therefore, if M. bovis is not properly identified or if there is resistance to other drugs, proper treatment may be hindered. Most laboratories in developing countries do not routinely perform mycobacterial cultures, and only a few laboratories can identify M. bovis. Therefore, HTBMb cases are believed to be underestimated. We report a large proportion of M. bovis isolates and an increasing isolation trend across time. We report a large proportion of M. bovis isolates from pulmonary samples, suggesting the possibility of human-to-human airborne transmission. Also, we showed that M. bovis isolates were more frequently resistant to streptomycin, perhaps as a result of antibiotic usage in cattle. This work underscores the need for identification to the species level, proper susceptibility testing, as well as a stricter control of bovine tuberculosis.