In this article we report the isolation, characterization and evaluation of in vitro anti-colon cancer activity of a new gingerol derivative, namely (S,E)-5-hydroxy-1-(4-hydroxy-3-methoxyphenyl)tetradec-8-en-3-one, (N6), and a known sesquiterpene (1,2-Dihydroxybisabola-3,10-diene,N7), isolated from Zingiber officinale rhizomes for the first time, together with other five known compounds; 6-gingerol (N1), 8-gingerol (N2), 10-gingerol (N3), 4-gingerol (N4) and 4`-O-methyl-6-gingerol (N5). The isolated compounds were identified using different spectroscopic techniques (1D and 2D-NMR, MS and IR). A modified chromatographic method was established and optimized to isolate the major gingerols (N1, N2, and N3) in grams.