Fusarium wilt of bananas (FWB) is seriously affecting the sustainable development of the banana industry and is caused by the devastating soil-borne fungusFusarium oxysporumf. sp.cubensetropical race 4 (Foc TR4). Biological control is a promising strategy for controlling Fusarium wilt in bananas. We previously identifiedStreptomyces hygroscopicussubsp.hygroscopicus5–4 with strong antifungal activity against the FWB. The most possible antimicrobial mechanism of strain 5–4 was explored using the metabolomics approach, light microscopy imaging, and transmission electron microscopy (TEM). The membrane integrity and ultrastructure of Foc TR4 was damaged after extract treatment, which was supported by the degradation of mycelium, soluble protein content, extracellular reducing sugar content, NADH oxidase activity, malondialdehyde content, mitochondrial membrane potential, and mitochondrial respiratory chain complex enzyme activity. The extracts of strain 5–4 cultivated at different times were characterized by a liquid chromatography–mass spectrometer (LC-MS). 647 known metabolites were detected in the extracts of strains 5–4. Hygromycin B, gluten exorphin B4, torvoside G, (z)-8-tetradecenal, piperitoside, sarmentosin, pubescenol, and other compounds were the main differential metabolites on fermentation culture for 7 days. Compared with strain 5–4 extracts, hygromycin B inhibited the mycelial growth of Foc TR4, and the EC50concentration was 7.4 μg/mL. These results showed that strain 5–4 could destroy the cell membrane of Foc TR4 to inhibit the mycelial growth, and hygromycin B may be the key antimicrobial active metabolite.Streptomyces hygroscopicussubsp.hygroscopicus5–4 might be a promising candidate strain to control the FWB and provide a scientific basis for the practical application of hygromycin B as a biological control agent.