The herbicidal efficacy and crop safety of Fusarium acuminatum strain HL-29, an endophytic fungus isolated from infected Amaranthus retroflexus in Qinghai Province, were evaluated. In vitro leaf assays demonstrated its pathogenicity against four broadleaf weeds, with efficacy ranked as follows: Elsholtzia densa = Senecio vulgaris = Chenopodium album > Malva verticillata. Pot trials further confirmed that the HL-29 fermentate caused 100% disease incidence in S. vulgaris, C. album, and E. densa. Notably, the strain showed no pathogenicity toward seven major local crops, indicating excellent selectivity. Scanning electron microscopy (SEM) revealed key morphological changes during the infection process on C. album leaves. These results establish F. acuminatum HL-29 as a promising biocontrol candidate against broadleaf weeds in the Qinghai–Tibet Plateau, providing a theoretical foundation for developing alpine-adapted mycoherbicides.