Background. Despite the recent advancement in diagnostic methods, the smear microscopy remains the gold standard for the diagnosis of pulmonary tuberculosis in high burden countries like Ghana. Notwithstanding, fluorescence staining technique provides a more efficient option for the detection of Mycobacterium tuberculosis positive smears. This study therefore aimed at assessing the diagnostic performance of fluorescence microscopy (FM) and Ziehl-Neelsen (ZN) staining techniques in the diagnosis of pulmonary tuberculosis. Methods. A comparative study was carried out on 100 patients who reported at the Out Patients Department (OPD) or the Directly Observed Therapy (DOT) center of the Kade Government Hospital and were suspected of having pulmonary tuberculosis. Two (2) sputum samples each were collected. This included one spot and one morning sample. The smears were prepared and stained with FM and ZN staining techniques. Xpert MTB/RIF assay was also performed. Results. Of the 200 samples analyzed, 71 (35.5%), 46 (23.0%), and 84 (42.0%) were positive for pulmonary tuberculosis when FM, ZN, and XPERT MTB/RIF assays were used, respectively. The mean reading time of FM was three times faster than the ZN technique with very good acceptance (1.5min: 4.6min). The sensitivity and specificity of fluorescent staining to that of XPERT MTB/RIF assay were 84.5% and 100%, respectively, while those of ZN staining were 54.8% and 100%, respectively. Conclusion. For a routine laboratory test in a resource-limited setting, our study has demonstrated that fluorescence staining technique is a more sensitive test for the diagnosis of pulmonary tuberculosis as compared to the conventional ZN technique.