Background The prompt diagnosis of pulmonary tuberculosis (PTB) remains a challenge in clinical practice. The present study aimed to optimize an algorithm for rapid diagnosis of PTB in a real-world setting. Methods 28,171 adult inpatients suspected of having PTB in China were retrospectively analyzed. Bronchoalveolar lavage fluid (BALF) and/or sputum were used for acid-fast bacilli (AFB) smear, Xpert MTB/RIF (Xpert), and culture. A positive mycobacterial culture was used as the reference standard. Peripheral blood mononuclear cells (PBMC) were used for T-SPOT.TB. We analyzed specimen types' effect on these assays' performance, determined the number of smears for diagnosing PTB, and evaluated the ability of these assays performed alone, or in combination, to diagnose PTB and nontuberculous mycobacteria (NTM) infections. Results Sputum and BALF showed moderate to substantial consistency when they were used for AFB smear or Xpert, with a higher positive detection rate by BALF. 3-4 smears had a higher sensitivity than 1-2 smears. Moreover, simultaneous combination of AFB and Xpert correctly identified 44/51 of AFB(+)/Xpert(+) and 6/7 of AFB(+)/Xpert(-) cases as PTB and NTM, respectively. Lastly, when combined with AFB/Xpert sequentially, T-SPOT showed limited roles in patients that were either AFB(+) or Xpert(+). However, T-SPOTMDC (manufacturer-defined cut-off) showed a high negative predicative value (99.1%) and suboptimal sensitivity (74.4%), and TBAg/PHA (ratio of Mycobacterium tuberculosis-specific antigens to phytohaemagglutinin spot-forming cells, which is a modified method calculating T-SPOT.TB assay results) >= 0.3 demonstrated a high specificity (95.7%) and a relatively low sensitivity (16.3%) in AFB(-)/Xpert(-) patients. Conclusions Concurrently performing AFB smear (at least 3 smears) and Xpert on sputum and/or BALF could aid in rapid diagnosis of PTB and NTM infections in a real-world high-burden setting. If available, BALF is preferred for both AFB smear and Xpert. Expanding this algorithm, PBMC T-SPOTMDC and TBAg/PHA ratios have a supplementary role for PTB diagnosis in AFB(-)/Xpert(-) patients (moderately ruling out PTB and ruling in PTB, respectively). Our findings may also inform policy makers' decisions regarding prevention and control of TB in a high burden setting.