The incidence and prevalence of nontuberculous mycobacterial disease is increasing due to enhanced clinician awareness and improved detection methods. The species identification using molecular microbiology techniques allows a better understanding of the differences in pathogenicity and treatment response. A 57-year-old man with a history of B-cell lymphoma in remission was transferred from the hematology department due to fever of unknown origin, night sweats and asthenia. The empirical antibiotic therapy was initiated with no clinical response, and he developed a subacute pneumonia, severe anemia and hepatosplenomegaly. After positive blood, bronchoalveolar lavage and bone marrow cultures, a disseminated Mycobacterium avium-intracellulare complex infection was diagnosed, and the patient began treatment with clarithromycin, rifabutin and ethambutol. Two weeks later, a fourth antibiotic was added, amikacin at first and then linezolid, with slow but gradual improvement. Due to amikacin-related severe kidney injury and linezolid-related severe myelosuppression, the fourth antibiotic was changed to moxifloxacin, which the patient tolerated. After 6 months of therapy, the sensitivity to the regimen was confirmed and the species was identified as Mycobacterium chimaera (MC), using the molecular genetic test GenoType NTM-DR. The blood and tissue cultures were negative after 4 months of therapy, and treatment was continued for 12 months. Although the infection was being treated successfully, the patient's B-cell lymphoma relapsed after 12 months and the patient died. This is a case report of a confirmed severe and disseminated MC infection in an immunocompromised patient using a molecular genetic test, successfully treated using a four-drug regimen.