Tuberculosis (TB) remains one of the major challenges to the global public health. The most powerful tools in any TB control program are prompt diagnosis and successful treatment of patients with active contagious disease. For almost 100 years the tuberculin skin test (TST) has been used to support the diagnosis of active and latent TB infection. The TST has a number of limitations, most notable low specificity in BCG vaccinated individuals due to cross-reactive components in PPD and the Mycobacterium bovis BCG vaccine strain and an intensive search for new and more specific diagnostic antigens has therefore been ongoing. The current diagnostic techniques utilize production of Interferon-gamma (IFN-γ) in response to novel M. tuberculosis (MTB) synthetic overlapping peptides mixtures to detect MTB infection. The aim of this study was to evaluate human immune responses to two novel Mycobacterium tuberculosis latency associated antigens Rv2659 Pepmix and Rv2660 Pepmix in comparison with ESAT-6 Pepmix. We compared the production of IFN-γ by ELISA following overnight stimulation with the antigens among the different groups of our study, TST negative healthy subjects (n = 16), TST positive healthy subjects (n = 16) and active pulmonary TB patients (n = 30). Our results showed that in TB patients, a positive IFN-γ response was observed to ESAT-6 by 73% of the donors, 47% responded to Rv2659 and 57% responded to Rv2660 when compared to TST negative controls. In conclusion, the ESAT-6 pepmix is recognized in a greater proportion of TB patients compared to Rv2659 and Rv2660, and levels of IFN-γ in response to ESAT-6 are higher than the levels observed in response to Rv2659 and Rv2660.