Background: Lippia origanoides H. B. K. (Verbenaceae) is an aromatic small shrub appreciated in the traditional systems of medicine. L. origanoides essential oil is an ingredient of commercial poultry feed products and its postdistillation residual biomass is an interesting source of bioactive compounds. During our search for the valorization of this residual biomass, supercritical-CO2(SC-CO2)extraction afforded a mixture that was subjected to an investigation of phytochemicals and of cytotoxicity, which was not reported previously. Objectives: The current study was designed to investigate the phytochemicals from the steam-distilled residual biomass of thymol- and carvacrol-rich L. origanoides chemotypes and to evaluate their in vitro cytotoxic activity. Materials and Methods: Steam-distilled aerial parts of L. origanoides chemotypes were extracted with SC-CO2to obtain a greenish-yellow extract with a strong aromatic odor. The SC-CO2extract was subjected to column chromatography, and the isolate obtained was screened in vitro for cytotoxicity against human normal embryonic kidney 293, MRC-5, THP-1, and XP4PA cell lines, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Results: A pentacyclic triterpene, friedelan-3-one (1) was isolated for the first time from L. origanoides chemotypes. The structure of the isolate was elucidated with spectroscopic (ultraviolet, infrared, mass spectra, and nuclear magnetic resonance) techniques. The in vitro cytotoxic activity of the isolated compound was determined. The results showed no significant results against the selected cell lines using the MTT assay. Conclusion: The main significance of the present study was to develop promising routes to utilize the residual biomass for value-addition. This is the first report of friedelan-3-one isolation from the genus Lippia.