Marine algae have been widely studied by natural products chemists in the search of antitumor agents and the development of new therapeutic agents against cancer. Extracts of the red alga Digenia simplex display a powerful antimitotic effect in cancer cell lines. Therefore, it is of extreme importance to determine what substances are responsible of this activity. In order to achieve this objective, the ethanol extract of Digenia simplex was subject to chromatographic fractionation guided by evaluation of antimitotic activity on the Jurkat cell line (human myeloid lymphoma). Additionally, the most active fraction obtained was tested for cytotoxicity. Normal phase column chromatography and preparatory thin layer chromatography were used for extract fractionation. Cytotoxicity was assessed by cellular viability methods, determining cellular viability by the exclusion of the vital dye trypan blue. A cytotoxic and antimitotic fraction of polar nature was obtained. This fraction behaved in a similar way to the antineoplasic drug Colcemid.