BACKGROUND: Nontuberculosis mycobacteria (NTM) are environmental pathogens that can cause pulmonary infections, especially among the immunocompromised population. There is limited research on healthcare professionals' knowledge of NTM infections in Ethiopia. This study aimed to evaluate healthcare professionals' knowledge about NTM infections at Debre Tabor Comprehensive Specialized Hospital (DTCSH). METHODS: A cross-sectional study was conducted from March 5 to July 30, 2024 at DTCSH to evaluate the knowledge of 292 healthcare professionals on NTM infections. A semi-structured questionnaire was used for data collection, and the data were analyzed using SPSS version 25. The study's findings were presented through texts, tables, and figures. RESULTS: Of all the participants, 160 (54.8%) were male, and the majority 153 (52.4%) were 36-45 years old. Nurses made up the largest professional group, 127 (43.5%). Approximately 59% of healthcare professionals showed good knowledge of NTM infections. In this study, a higher percentage of female [92 (69.7%)] participants demonstrated good knowledge compared to males [83 out of 160 males (51.9%)]. Similarly, 35 doctors (89.7%) and 80 nurses (63%) demonstrated favorable knowledge. Only 52.4% could distinguish between NTM and Mycobacterium tuberculosis infections. Notably, 77.4% recognized that NTM infections are more common in immunocompromised individuals, yet only 20.5% knew that NTM species are not typically treated with anti-tuberculosis drugs. CONCLUSION: The knowledge gaps among healthcare professionals regarding NTM infections necessitate focused educational initiatives to improve their ability to manage these infections. The focused training is essential for enhancing the management and identification of NTM infections.