To evaluate a one-tube nested PCR-based analysis of urine for diagnosing pulmonary tuberculosis (PTB) and extrapulmonary tuberculosis (EPTB) in Bobo-Dioulasso, Burkina Faso, a prospective analysis of urine samples from HIV- and non-HIV-infected adults with PTB and EPTB (case patients) and with pathology other than tuberculosis (TB) (control patients) was performed. Three groups of patients were classified as microbiological-positive and -negative PTB and EPTB on the basis of clinical signs and microbiological results. Urine from patients was analysed using the DNA extraction and Sechi's methods, both modified, for the detection of Mycobacterium tuberculosis. The sensitivity, specificity, positive predictive value and negative predictive value were calculated. The sensitivity of the test for the microbiological-positive PTB, microbiological-negative PTB and EPTB was 40.5 % (88/217), 66.7 % (20/30) and 57.1 % (48/84), respectively. The specificity was 98.2 %. Differences were observed in the two populations infected and not infected by HIV. This method is not appropriate for detection of new TB cases in the routine laboratory, but it can be useful for cases where the clinical and bacteriological diagnosis of TB is not conclusive.