The present work analyzed the antibacterial, cytotoxic, and antidiabetic activities of endophytic Diaporthe eres (SPEF004). The profiling of bioactive molecules was determined using GC-MS analysis and these molecules were docked with breast cancer-related heat shock protein (HSP90). Among the extracts, FEAE (Fungal ethyl acetate extract) showed zone of inhibition in the range of 9.06-27.5 mm against Staphylococcusaureus, Escherichia coli,Salmonella enterica, Bacillus cereus, and Listeria monocytogenes. The FEAE showed DPPH (61.53 +/- 1.47 %), ABTS (85.62 +/- 2.48 %) scavenging property, and inhibitory activity of 41.11 +/- 1.52 % for alpha amylase and 13.28 +/- 0.94 % for alpha glucosidase at the concentration of 100 mu g/mL. Interestingly, FEAE was found to be not toxic to mouse fibroblast (NIH3T3) cells, but, toxic to human breast cancer cell line (MDA MB231) through the damaging nucleus at an IC50 value of 27.5 +/- 0.25 mu g/mL. Furthermore, pyrrolidine-5-one, 2-[3-hydroxypropyl] derived from Diaporthe sp., exhibited a strong binding affinity score (-5.4 kcal/mol) with HSP90 through the interaction of hydrogen bonds, and it is expected to regulate the apoptosis in breast cancer cells. This work proved the bioactivities of the novel compounds identified from Diaporthe sp. (SPEF004) for the possible development of pharmaceutically viable agents.