Aim: The aim of this study was to assess the effectiveness of novel biomarkers in the pathological diagnosis of pulmonary tuberculosis (TB) and compare their diagnostic performance with conventional methods such as sputum smear microscopy, GeneXpert MTB/RIF, and chest X-ray. Material and Methods: This prospective cohort study included 80 patients diagnosed with suspected pulmonary tuberculosis at a tertiary care hospital. The study involved clinical evaluation, sputum collection for microscopy and GeneXpert MTB/RIF testing, chest X-ray, and novel biomarker assays (TNF-α, IL-6, IFN-γ, and MCP-1). Statistical analysis was performed using SPSS, and the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and ROC curve analysis were used to assess diagnostic performance. Results: The study found that sputum smear microscopy had a sensitivity of 40% and specificity of 95%, while GeneXpert MTB/RIF had superior performance with 85% sensitivity and 90% specificity. Novel biomarkers, particularly IFN-γ (91% sensitivity, 75% specificity), IL-6 (90% sensitivity, 68% specificity), and TNF-α (85% sensitivity, 70% specificity), demonstrated promising diagnostic accuracy. ROC curve analysis showed high AUC for IFN-γ (0.89) and IL-6 (0.88), supporting their potential as diagnostic adjuncts. Conclusion: This study highlights that novel biomarkers such as IFN-γ, IL-6, and TNF-α exhibit strong diagnostic performance for pulmonary tuberculosis and could complement traditional diagnostic methods, particularly in resource-limited settings where conventional methods have limitations.