Non-tuberculous mycobacteria comprise a group of more than 190 species with variable pathogenic potential. Challenges in managing suspected infections include difficulty distinguishing contamination from infection, low sensitivity and slow turnaround of traditional microbiological methods, and empirical, prolonged treatment regimens. Molecular methods have expanded the capacity to identify these microorganisms. The reported case is of a 76-year-old man with type 2 diabetes mellitus, with progressive right otalgia for 6 months associated with otorrhea, aural fullness sensation, and ipsilateral hearing loss. He was treated with multiple empirical topical and oral antibiotic regimens during this period. After hospital admission, necrotizing otitis externa and right otomastoiditis (OM) were evidenced on CT, and empirical therapy with cefepime was instituted for 9 weeks. In culture of material obtained after myringotomy during this admission, Pseudomonas aeruginosa susceptible to cefepime was isolated. He was discharged after completing treatment and returned one month later with worsening. At this time, mastoidectomy was performed with collection of intraoperative materials for culture, initially interpreted as therapeutic failure, and a new course of cefepime was started for another 6 weeks. During this period, mycobacterial growth occurred in appropriate culture (Lowenstein-Jensen medium) from a bone fragment, but it was not identified by mass spectrometry. Molecular rapid test for tuberculosis (TRM-TB) from the operative material was negative. The isolate was then submitted to PCR amplification of the rpoB gene, confirming identification as Mycobacterium arupensis. At that time, the patient still had active OM demonstrated clinically and radiologically, and empirical treatment with linezolid, clarithromycin, and ethambutol was initiated based on literature review. Despite treatment, the patient developed decreased level of consciousness and died after 2 months. The first description of disease associated with M. arupensis was in a case of tenosynovitis (TS) from a tendon isolate in 2006. TS after traumatic injuries is the most commonly described infection related to this species, but osteoarticular, pulmonary, and disseminated infections in immunocompromised individuals have been described. In our literature review, we found no other cases of chronic OM due to M. arupensis reported in the literature.