Commiphora habessinica resin is used traditionally to treat various diseases in Ethiopia. In this study the cytotoxic effects of chloroform fraction and cholesterol and lathosterol isolated from the chloroform fraction of the resin of C. habessinica were evaluated. In the cytotoxicity assay on A549, A2780, MIA-Paca-2, and SNU-638 cell lines, the chloroform fraction showed cytotoxicity ranging from 0.77-3.35 ?g/ml. The chloroform fraction significantly inhibited cell proliferation of A549, A2780, MIA-PaCa-2 and SNU-638, with dose-dependent relation in vitro. The chloroform fraction was more sensitive and has a strongest net growth as percent control effects on A549 cell lines. A mixture of cholesterol : lathosterol (47.9% : 52.1%) exhibited a moderate cytotoxicity which is greater than the individual compound towards A549 and A2780 with IC50 of 13.77 and 20.36 ?g/ml, respectively that might be due to synergetic effect. The GC-MS analysis of chloroform fraction of the resin showed presence of pentacyclic triterpenes as major component (62.98%), sesquiterpene (4.27%), phytosterols (1.53%) and others in trace amount. The isolated compounds from the chloroform fraction were analysed by spectroscopic techniques such as NMR and MS.