Ethyl acetate extracts obtained from culture of endophytic fungi Aspergillus sp isolated from Piper crocatum Ruiz and Pav, have been shown to possess cytotoxic activity against T47D breast cancer cells. Investigations were here conducted to determine bioactive compounds responsible for the activity. Bioassay guided fractionation was employed to obtain active compounds. Structure elucidation was performed based on analysis of LC-MS, 1H-NMR, 13C-NMR, COSY, DEPT, HMQC, HMBC data. Cytotoxity assays were conducted in 96 well plates against T47D and Vero cell lines. Bioassay guided isolation and chemical investigation led to the isolation of pyrophen, a 4-methoxy-6-(1'-acetamido-2'-phenylethyl)-2H-pyran-2-one. Further analysis of its activity against T47D and Vero cells showed an ability to inhibit the growth of T47D cells with IC50 values of 9.2 μg/mL but less cytotoxicity to Vero cells with an IC50 of 109 μg/mL. This compound at a concentration of 400 ng/mL induced S-phase arrest in T47D cells.