Emergence of multidrug-resistant (MDR) Mycobacterium tuberculosis isolates constitutes a threat to public health worldwide. This study aimed at acquiring first epidemiological data for Guyana. Thirty-six M. tuberculosis isolates from patients of the Georgetown Chest Clinic were subjected to susceptibility testing on solid agar and in broth media. Resistance to at least one first-line drug was observed in 8 (22.2%, 95% confidence interval 8.3–36.1%) and simultaneous resistance to rifampicin and isoniazid (MDR) in 4 (11.1%, 95% confidence interval 0.6–21.6%) of the 36 isolates. The risk of infection with resistant isolates was significantly related to earlier antituberculosis therapy (P = 0.040). These data indicate a high proportion of resistant M. tuberculosis isolates in Guyana and call for the implementation of control strategies based on an improved laboratory diagnosis of TB.