Mycobacterium genavense, a nontuberculous Mycobacterium, is found in immunosuppressed patients, particularly in those with HIV. Mycobacterium genavense incubation under standard culture conditions is difficult, and its identification is challenging using routine culture methods. Herein, we report the case of a 40-year-old Japanese man with HIV presenting with disseminated M. genavense infection. An analysis using an automated blood culture system did not show positive signals during 6 weeks of incubation. However, an acid-fast bacilli smear of his blood sample was positive for the bacterium. Mycobacterium genavense was identified using sequencing analysis, targeting the heat shock protein 65 gene. The patient recovered from the infection, following antibiotic therapy for 18 months. Under suspicion of disseminated M. genavense infection and the absence of bacterial growth in blood culture samples, an acid-fast bacilli smear test of the sample may be useful for timely diagnosis.