Bright field microscopy using Ziehl-Neelsen (ZN) stained smear has been the major diagnostic technique for the diagnosis of pulmonary tuberculosis and treatment initiation in resource limited settings. However, it has its own limitation in terms of its sensitivity. Various reports have shown superior sensitivity of light emitting-diode fluorescent microscopy (LED-FM) and comparable specificity to bright field microscopy. The aim of the study was to compare the results of Auramine O-stained sputum smears by LED-FM against Ziehl-Neelsen stained sputum smears and JD-TB-Antigen test using TB culture as a reference test. A cross-sectional study was conducted in Southern Nations and Nationality People Regions (SNNPR) of Ethiopia from September 2013 to November 2014. A total of 248 sputum samples were collected from different Health Centers and Hospitals of the study area, analyzed by LED-FM and ZN stained bright field microscopy. The sensitivity, specificity, positive and negative predictive value were 66.12%, 95.28%, 93.02% and 74.69% for ZN stained microscopy; 81.82%, 93.70%, 92.50% and 98.35% for LED–FM, and 58.33%, 63.27%, 53.85% and 67.39% for JD-TB-Antigen test, respectively. The correlation between LED-FM, ZN and culture have shown statistically significant (p<0.001). The sensitivity of LED-FM is better than ZN-stained bright field microscopy. It is also easy to perform, save time and better choice for sputum microscopic examination. The performance of JD-TB-Antigen test was very low for the diagnosis of tuberculosis in sputum specimens. Therefore, further study should be done to use JD-TB-Ag test for diagnosis of pulmonary tuberculosis