Tuberculosis (TB) caused by acid-fast bacilli, specifically Mycobacterium tuberculosis, is indeed one of the most serious infectious diseases globally. Early detection, identification, and treatment of active tuberculosis reduce the risk of infection. The key challenge in controlling the burden of illness is the absence of a reliable diagnostic method. The primary objective of this research is to evaluate the role of Truenat in comparison with the Ziehl-Neelsen technique and fluorescent microscopy in patients suspected of having Koch’s bacillus, and to determine the prevalence of rifampicin resistance among the positive cases. A prospective cross-sectional study was carried out in our laboratory for one year. Overall, 229 samples were tested using all three methods, 60% were from males and 40% from females, with the most severely affected individuals aged 21-45 years (60.6%). The positivity of Ziehl-Neelsen stain was 22.3% (n = 51), fluorescent stain was 24.0% (n = 55), and Truenat was 26.6% (n = 61), respectively. Rifampicin resistance was identified in one positive case. The conventional methods were compared to the molecular technique, considering it to be the gold standard for assessing sensitivity, specificity, positive predictive value, and negative predictive value. The recorded value was quantitatively analysed, with a P-value below 0.05 being statistically significant. Truenat has demonstrated itself to be a more effective method when compared to conventional techniques. Among the microscopy techniques, the fluorescence stain is found to be more accurate and rapid than the Ziehl-Neelsen stain.