Leprosy is an infectious and chronic dermatoneurological disease, considered the most frequent cause of physical disabilities worldwide. It remains a major challenge for public health in Brazil, which is classified as a country with a high burden of the disease and ranks second in number of cases among all nations. Laboratory tests are important tools to complement the clinical-epidemiological evaluation, and molecular techniques emerged in the hope of reducing the limitations existing in the laboratory diagnosis of leprosy. We aimed to evaluate the accuracy of real-time polymerase chain reaction (qPCR) in the detection of Mycobacterium leprae in direct intradermal scrapings (swabs) and smears from bacilloscopy slides. This is an observational and cross-sectional study to evaluate diagnostic tests, which used bacilloscopy as the gold standard. Participants with epidemiological and/or clinical suspicion of leprosy from the northeastern region of Minas Gerais, referred to the Macroregional Laboratory of Teófilo Otoni for collection of intradermal scrapings from the earlobes, right elbow, left elbow or suspicious lesion were included. Bacilloscopy was performed according to the Technical Procedures Guide of the Ministry of Health and the DNA samples, after extraction and quantification, were subjected to qPCR with primers specific for the RLEP region of Mycobacterium leprae. The study obtained the number of 45 samples collected from suspected cases of leprosy in the period between May/2022 and February/2023. Descriptive, comparative and accuracy statistical analyses were performed using Minitab 17 and GraphPad Prism version 6.00 software. The sensitivity of qPCR on direct intradermal scraping samples (swabs) and smear slides, compared with bacilloscopy, was 100%. The specificity was higher for qPCR on swab samples (64.3%) than on smear slides (53.8%). The performance of qPCR on intradermal scraping swab samples and smear slides indicated accuracies of 82.10 and 76.9%, respectively. The results of the comparison of the proportions of qPCR positivity of the two samples showed a statistically significant association (P-value = 0.005), with a degree of agreement of 55% (Kappa = 0.55). Taken together, the results of the performance and comparative analyses of qPCR on intradermal scraping swabs and smear slides showed good accuracy in relation to bacilloscopy and moderate agreement between these different qPCR strategies.