Tuberculosis is a zoonotic infectious disease, caused by bacteria of the tuberculosis complex. Mycobacterium bovis causes tuberculosis in water buffaloes (Bubalus bubalis), and it can also infect other domestic animals, wild fauna, and humans. The diagnosis of bovine tuberculosis (bTB) through intradermal injections is challenging, and to understand the behaviour of other diagnostic tests is crucial. The aim of this research is to analyse three diagnostic tests against bTB in water buffaloes with positive test DPP. Different diagnostic tests were tested in 50 buffaloes diagnosed with bTB Cervical Comparative Test, from the Colombian lower tropic. Lesions compatible with bTB in 26 buffaloes with a positive DPP ; Four out of 2 samples of Mycobacterium bovis in DPP-positive buffaloes were isolated and confirmed positively in tissues using PCR-HRM, three buffaloes showed granulomatous lesions in histological analyses with positive microbiological isolation; 17 DPP-positive buffaloes had a positive PCR-HRM test and nine of these buffaloes showed no histological findings compatible with bTB, leading to purely molecular diagnosis. Evidence in histological, microbiological, and molecular findings in DPP is positive for the water buffaloes. None of the complementary tests showed 100% concordance with the intradermal results obtained with the Cervical Comparative Test for bTB.