This study aimed to extract bioactive secondary metabolites, determine volatile organic compounds, and polyphenols using GC/MS and LC/MS, and assess their total phenolic content, antimicrobial, antioxidant, and cytotoxic properties. The chemical analysis of S. cerevisiae NRLL Y‐12633 ethyl acetate extract revealed eight volatile organic components, whereas the main component was phenyl ethyl alcohol (77.39 %). On the other hand, nineteen volatile organic compounds were identified in the ethyl acetate extract of S. cerevisiae NRRL Y‐1089, whereas the most critical component was 2, 3‐butanediol (60.74 %). The most abundant phenolic acids were syringic acid (25476.46 ng/mL), and cinnamic acid (5163.76 ng/mL) in the ethyl acetate extracts of S. cerevisiae NRLL Y‐12633 and S. cerevisiae NRRL Y‐1089 respectively. Flavonoid, naringenin, was also found in the ethyl acetate extract of S. cerevisiae NRLL Y‐12633, whereas daidzein and rutin were detected in the ethyl acetate extract of S. cerevisiae NRRL Y‐1089. The total phenolic content of S. cerevisiae NRRL Y‐12633 ethyl acetate extract was found to be greater than that of S. cerevisiae NRRL Y‐1089 ethyl acetate extract. Data revealed that S. cerevisiae ethyl acetate extracts displayed antioxidant and antimicrobial activity, whereas only the ethyl acetate extract of S. cerevisiae NRRL Y‐1089 showed anticancer activity.