Abstract: Streptomyces hygroscopicus HU216, isolated from the sediment of Taihu Lake, exhibits broad-spectrum antifungal activity against plant pathogenic fungi. To further explore its active metabolites, the fermentation broth of strain HU216 underwent fractionation and purification through silica gel chromatography, gel column chromatography, and preparative high-performance liquid chromatography (HPLC). Six pure compounds were isolated and their structures were determined by mass spectrometry (MS) and nuclear magnetic resonance spectroscopy (NMR, including 1D and 2D NMR). These compounds were identified as ascomycin (1), 3-(1-hydroxypropyl)phenol (2), abierixin (3), (2E,4Z)-8-hydroxydeca-2,4-dienoic acid (4), cuevaene A (5), and 29-O-methylnigericin (6). The antifungal activities of these compounds were evaluated against six plant pathogenic fungi. The results showed that six compounds exhibited significant antifungal activity at a concentration of 60 μg/tablet. Specifically, compounds 1, 2, and 3 showed inhibition rates of 85.83%, 75.74%, and 70.15% against Botrytis cinerea; 89.38%, 76.63%, and 62.25% against Valsa mali; and 63.40%, 64.22%, and 58.50% against Bipolaris maydis, respectively. The antifungal activity of these compounds was comparable to that of the positive control tebuconazole, demonstrating effective antifungal properties. The control efficacy tests of compounds 1-3 on tomato fruits inoculated with gray mold demonstrated that these compounds effectively protect tomato fruits from gray mold infection, showing potential for development into new agricultural antibiotics.