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Background Rapid diagnosis is essential to decrease the morbidity and mortality of Pott’s disease. The bacteriological methods are time-consuming or insensitive. Polymerase chain reaction (PCR) provides a rapid diagnostic tool and hope for early diagnosis of this disease. The aim of this study was to compare and assess of a rapid and effective method among diagnostic battery (Ziehl–Neelsen (ZN) microscopy, BACTEC culture and PCR) of Pott’s disease. Materials and Methods Sixty-five specimens from clinico-radiological suspected cases of Pott’s disease were included in this study. They were processed for ZN microscopy, BACTEC culture, and PCR IS6110. The tests tool’s efficiency, positive agreement K c (Kappa coefficient), and significance level (P value) were calculated for correlation between PCR and performed tests. Results The PCR sensitivity reached to 96% and 46.3% among positive and negative specimens on ZN microscopy. Further, 94% and 36.4% sensitivity were found among positive and negative specimens by BACTEC culture. The total 38 (58.5%) specimens were detected either ZN microscopy or by BACTEC culture. Thus, the overall sensitivity and specificity of PCR were 95% and 74.1%. The kappa coefficient and P value, calculated for PCR against BACTEC culture and combined results of performed bacteriological tests were (K c=0.60, (P<0.001)) and (K c=0.70, (P<0.001)), respectively. Above statistical relations showed a fair agreement with significant differences. Conclusion The PCR IS6110 may be useful in rapid detection of clinico-radiological suspected cases of Pott’s disease and those that are negative with bacteriological methods.