With the rise of drug-resistant diseases, there is a growing demand for innovative medicines that do not rely on chemosynthetic medications. It has been believed that medicinal plants provide an ideal habitat for various bioactive microbial communities. The medicinal plants endophytes Chaetomium interruptum and Chaetomium laterale were examined for their antioxidant, antiviral, cytotoxic, and anticancer properties in this study. Chemigraphic separation and identification of metabolic components were employed in the chromatographic analysis of Chaetomium interruptum ethyl acetate and petroleum ether extracts. Phytochemical assessment of the extracted metabolites indicated the presence of phenolics, esters, alcohols, and hydrocarbons in both extracts. The key fundamental isolated compounds were nine ester compounds, acetyl tributyl citrate (ATBC) of (35.29 and 45.91%), hexadecanol of (8.35%), phenol compounds of (18%), 2,4-ditert-butylphrenol of (13.40%), and alcohols of (8.3%). The fungal extracts exhibited radical-scavenging capacity, antiproliferative effectivity against three human carcinoma cell lines, and minimal toxicity towards normal cells. Cellular shape and morphology alterations were captured in the hepatocellular carcinoma cell line after being treated with C. interruptum ethyl acetate extract. In vitro, mild to moderate cytopathic inhibition effectivity of both fungal extracts against Herpes Simplex Virus, type-1 was detected. Genus Chaetomium is still a valuable source of species in biomedical applications.