Four new metabolites, including a cyclic tetrapeptide asperhiratide A (1), an ecdysteroid derivative asperhirasterone A (2), a symmetrical thioether asperhirathioether A (3), and a sesquiterpene lactone asperhiranone A (4), were isolated and identified from the soft coral-derived fungus Aspergillus hiratsukae SCSIO 5Bn1003 together with 11 known compounds. Their structures were elucidated by spectroscopic analysis, X-ray diffraction analysis, and electronic circular dichroism. In addition, the absolute configuration of 1 was determined by the Mosher ester technique and analysis of the acid hydrolysates using a chiral-phase HPLC column. Among all the compounds, 7 and 9 showed medium cytotoxic activities against four tumor cell lines (SF-268, HepG-2, MCF-7, and A549) with IC50 values ranging from 26.39 ± 1.24 to 50.25 ± 0.54 µM. Meanwhile, compounds 7 and 9 strongly inhibited α-glucosidase activities with IC50 values of 35.73 ± 3.94, 22.00 ± 2.45 µM,7-9 showed significant antibacterial activities against Bacillus subtilis with MIC values of 10.26 ± 0.76µM, 17.0 ± 1.25µM, 5.30 ± 0.29 µM, respectively. Besides, asperhiratide A (1) were evaluated for anti-angiogenic activities in an in vivo zebrafish model, which showed a weak inhibitory effect on intersegmental vessel (ISV) formation. This is the first report of chemical and bioactive investigation of A. hiratsukae.