Background: Despite the advent of newer drugs, modern diagnostic techniques and improvement in management strategies, India continues to be the leader in tuberculosis burden. Several methods for diagnosis exist, such as light microscopy, fluorescent microscopy, solid culture, liquid culture (Mycobacterium growth indicator tube [MGIT] culture) to molecular methods. However, each of these methods have variable sensitivity and specificity. Very few studies have been done evaluating the role of TrueNAT with fluorescent microscopy, thereby evaluating the performance of a designated microscopy center under the national tuberculosis elimination programme. Methods and Materials: This is a retrospective observational study performed in a designated microscopy center in Santhiram Medical Center, Nandyal. This is to compare the fluorescence microscopy and TrueNAT provided as part of the National Tuberculosis Elimination programme. A total of 342 patients underwent testing at this center and were diagnosed with tuberculosis. Results:Majority of the study participants were males, smokers, in the age groups of 31-40 years. The mean age of males was found to be higher than that of females. (37.64 vs 32.39 years). 89.42% of patients were diagnosed on fluorescent microscopy, while 94.23% were diagnosed with TrueNAT. Sensitivity of TrueNAT was 91.61%, specificity was 87.80% and accuracy was 90.76%. Conclusion: TrueNAT appears to be superior to Fluorescent microscopy in a setting of a designated microscopy center.This can be used for rapid diagnosis, with high sensitivity with specificity when compared to the traditionally accepted fluorescent microscopy.