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Diagnosis of bovine tuberculosis (bTB) may be confounded by immunological cross-reactivity to <i>Mycobacterium bovis</i> antigens when animals are sensitised by certain nontuberculous mycobacteria (NTMs). Therefore, this study aimed to investigate NTM species diversity in African buffalo (<i>Syncerus caffer</i>) respiratory secretions and tissue samples, using a combination of novel molecular tools. Oronasal swabs were collected opportunistically from 120 immobilised buffaloes in historically bTB-free herds. In addition, bronchoalveolar lavage fluid (BALF; <i>n</i> = 10) and tissue samples (<i>n</i> = 19) were obtained during post-mortem examination. Mycobacterial species were identified directly from oronasal swab samples using the Xpert MTB/RIF Ultra qPCR (14/120 positive) and GenoType CM<i>direct</i> (104/120 positive). In addition, all samples underwent mycobacterial culture, and PCRs targeting <i>hsp</i>65 and <i>rpoB</i> were performed. Overall, 55 NTM species were identified in 36 mycobacterial culture-positive swab samples with presence of <i>esat</i>-6 or <i>cfp</i>-10 detected in 20 of 36 isolates. The predominant species were <i>M. avium</i> complex and <i>M. komanii.</i> Nontuberculous mycobacteria were also isolated from 6 of 10 culture-positive BALF and 4 of 19 culture-positive tissue samples. Our findings demonstrate that there is a high diversity of NTMs present in buffaloes, and further investigation should determine their role in confounding bTB diagnosis in this species.