Previous studies have demonstrated that cells from both multi-drug-resistant tuberculosis (MDR-TB) and non-tuberculous mycobacteria (NTM) patients respond poorly to mycobacterial antigens in vitro. In the present study, we compared the in vitro response of cells isolated from sensitive TB (NR-TB)-, MDR-TB- and NTM-infected patients. Analysis of T cell phenotype ex vivo revealed that both MDR-TB and NTM patients present an increased percentage of CD4+CD25+- forkhead box protein 3 (FoxP3)+ and CD4+CD25+CD127− regulatory T (Treg) cells when compared to NR-TB. Increased numbers of Treg cells and interleukin (IL)-10 serum levels were detected in MDR-TB, whereas elevated serum transforming growth factor (TGF)-β was found in the NTM group. Cells of MDR-TB patients stimulated with early secretory antigenic target (ESAT)-6, but not purified protein derivative (PPD), showed a lower frequency of CD4+/interferon (IFN)-γ+ T cells and enhanced CD4+CD25+FoxP3+, CD4+CD25+CD127− and CD4+CD25+IL-10+ T cell population. In addition, increased IL-10 secretion was observed in cultured MDR-TB cells following ESAT-6 stimulation, but not in NR-TB or NTM patients. In vitro blockade of IL-10 or IL-10Rα decreased the CD4+CD25+FoxP3+ frequencies induced by ESAT-6 in MDR-TB, suggesting a role of IL-10 on impaired IFN-γ responses seen in MDR-TB. Depletion of CD4+CD25+ T lymphocytes restored the capacity of MDR-TB T cells to respond to ESAT-6 in vitro, which suggests a potential role for Treg/T regulatory 1 cells in the pathogenesis of MDR-TB. Together, our results indicate that although the similarities in chronicity, NTM- and MDR-TB-impaired antigenic responses involve different mechanisms.