Plant pathogenic fungi seriously threaten global crop production, and endophytic fungi are promising reservoirs of bioactive antifungal metabolites. Three undescribed polyester derivatives, talapolyesters I–K (1–3), along with thirteen known compounds including 15G256ω (4), 15G256ι (5), 15G256α (6), talapolyester E (7), 15G256α-1 (8), 15G256α-2 (9), 15G256α-2-me (10), 15G256ν (11), ES-242-3 (12), dongtinganthracene A (13), penicillide (14), 3-methyl-6-hydroxy-8-methoxy-3,4-dihydroisocoumarin (15), and (R)-6-hydroxymellein (16), were isolated from the endophytic fungus Talaromyces striatoconidius WI-F2. Their chemical structures were elucidated comprehensively using NMR and MS spectroscopic analyses, combined with alkaline hydrolysis. Compounds 4–8 exhibited in vitro promising antifungal activity against Fusarium oxysporum f. sp. cubense, with half-maximal inhibitory concentration (IC₅₀) values of 9.72, 21.07, 7.89, 8.91 and 9.65 μg/mL respectively, all markedly lower than that of ketoconazole (46.23 μg/mL). Molecular docking simulations further validated the observed antifungal activity, with binding energies ranging from −9.01 to −12.25 kcal/mol, indicative of stronger binding affinity compared with benzamidine (−6.80 kcal/mol). This study offers new clues for research on antifungal agents.