Objective To analyze the clinical characteristics of co-infection of Talaromyces marneffei (TM) and non-tuberculous Mycobacterium (NTM) in human immunodeficiency virus (HIV)-negative patients. Methods Clinical data of 8 HIV-negative patients with co-infection of TM and NTM in a hospital from 2019 to 2022 were co-llected. Clinical manifestations, auxiliary examination, treatment and prognosis were retrospectively analyzed. Results Among the 8 patients, 5 were females and 3 were males, with an average age of (52.25±12.31) years old. All patients presented TM and NTM disseminated infection. The major involved organs were lung (100%), lymph nodes (87.5%), and skin (75.0%). Clinical symptoms mainly included cough and expectoration (87.5%), fatigue (62.5%), joint and lumbosacral pains (62.5%), fever (50.0%), as well as skin and soft tissue abscess (50.0%), etc. Anti-interferon-γ (INF-γ) autoantibodies were detected in 4 patients and the results were positive. All 8 patients (100%) had pulmonary lesions, with chest CT mainly showing spots, patches, and striped shadows in both lungs. Among them, 7 cases (87.5%) had increased and enlarged mediastinal lymph nodes, 4 cases (50.0%) had pleural thickening and pleural effusion, 2 cases each (25.0% for each) were accompanied by pulmonary mass shadows, bronchial stenosis, as well as increased and enlarged hilar lymph nodes. One case each (12.5% for each) had pulmonary cavity formation, bronchiectasis, and pericardial effusion. Conclusion The co-infection of TM and NTM in non-HIV patients presents disseminated infection, with multiple clinical symptoms. Chest imaging shows a wide variety of pulmonary lesions. It is prone to miss diagnosis in clinic, and the effect is not ideal after treatment for single pathogen infection.