The occurrence of mycobacterial infections in different hosts and their implication as obligate or opportunistic pathogens remain mainly unclear. In addition to the well-known pathogenic members of the Mycobacterium tuberculosis - complex (MTBC), over 180 nontuberculous mycobacteria (NTM) species have been described. Although the large majority of the NTM are assumed to be non-pathogenic to most individuals, an increasing trend in NTM infections has been observed over the last decades. The reasons of such augmentation are probably more than one: improved laboratory diagnostics, an increasing number of immunocompromised patients and individuals with lung damage are some of the possible aspects. Mandibular lymph nodes of 176 hunted wild boars from the pre-Alpine region of Canton Ticino, Switzerland, were collected. Following gross inspection, each lymph node was subjected to culture and to an IS6110 based real-time PCR specific for MTBC members. Histology was performed of a selection of lymph nodes presenting gross visible lesions. Moreover, accuracy of matrix-assisted laser desorption ionization–time of flight (MALDI-TOF) mass spectrometry species identification was compared with sequence analysis of a combination of housekeeping genes. Mycobacteria of the MTBC were detected in five out of 176 wild boars (2.8%; CI95% 1.2 - 6.5) and were all confirmed to be Mycobacterium microti by molecular methods. In addition, based on the examined lymph nodes, NTM were detected in 57.4% (CI95% 50.0 – 64.5) of the wild boars originating from the study area. The 111 isolates belonged to 24 known species and three potentially undescribed Mycobacterium species. M. avium subsp. hominissuis thereby predominated (22.5%) and was found in lymph nodes with and without macroscopic changes. Overall, the present findings show that, with the exception of undescribed Mycobacterium species where identification was not possible (3.6%; 4/111), MALDI-TOF had a high concordance rate (90.1%; 100/111 isolates) to the sequence based reference method.