RESUMEN. Empleando las lineas celulares tumorales MCF-7, MKN-45, HT-29, SiHa, HEp-2, HeLa y U937 se evaluo la actividad citotoxica in vitrode las fracciones acuosa (FW), butanolica (FB), metanolica (FM) y en diclorometano (FD) obtenidas del extracto etanolico total de Acnistus arborescens. Se encontro que FM, FB y FD presentan una actividad promisoria sobre todas las lineas, destacandose el efecto citotoxico de FD, con valores de concentracion letal 50 (CL 50 ) entre 18,5 ± 1,3 y 7,4 ± 0,6 µg/mL. El analisis fitoquimico preliminar, realizado por diferentes metodos cromatograficos, permitio determinar la presencia de esteroides y terpenos en todas las fracciones, witanolidos en FD, esteres y acidos grasos en la fraccion hexanica (FH) y flavonoides en FB, estos ultimos no descritos antes para esta especie. Los resultados confirman que Acnistus arborescensademas de los witanolidos, es fuente de otros metabolitos de interes farmacologico. SUMMARY. “Cytotoxic Activity and Phytochemical Analysis of Fractions obtained from Total Ethanolic Extract of Acnistus arborescens”. In vitrocytotoxic activity of butanolic (BF), methanolic (MF), and dicloromethane (DF) fractions obtained from the total ethanolic extract of Acnistus arborescenswere evaluated against MCF-7, MKN-45, HT-29, SiHa, HEp-2, HeLa and U937 tumoral cell lines. MF, BF and DF showed a promising cytotoxic activity against all cell lines, with DF highlighted by its low lethal concentration 50 (LC 50 ), values between 18,5 ± 1,3 and 7,4 ± 0,6 µg/mL. Preliminary phytochemical analysis, carried out by different chromatographic methods, revealed the presence of steroids and triterpenoids in all fractions, withanolides in DF, esthers and fatty acids in the hexanic fraction (HF) and flavonoids in BF. Flavonoids have not been described before for this species. The results confirm that Acnistus arborescensis a source of withanolides and other compounds with potential pharmacological application, besides withanolides.