Bidens pilosa L. is a plant considered medicinal by some South American cultures. It contains polyacetylenes which may be the constituents responsible for its antitumor activity. Extracts obtained by hydroethanol maceration (HCE) and supercritical fluid extraction (SFE) were monitored for antitumor activity and the presence of polyacetylenes in the constitution. Both extracts killed concentration-dependently the MCF-7 cells in culture, although the SFE extract presented superior cytotoxic activity. The SFE presented IC50=437 (428–446)μg/mL in 24h of incubation, decreasing to IC50=291 (282–299)μg/mL at 48h. The HCE started causing DNA cleavage at 160μg/mL while the SFE extract started at 40μg/mL, a concentration enough to initiate the in vitro cleavage. The presence of polyacetylenes as the major compounds in SFE was confirmed by TL chromatography combined with UV–vis analyses. Ehrlich ascites carcinoma-bearing mice were used for the antitumor study. Animals were divided in five groups: normal, negative control, positive control (Doxorubicin 0.06mg/kg), test group HCE and test group SFE (100mg/kg b.w. per day). After 9 days of treatment, 50% of randomly chosen animals from each group were sacrificed for the study. The parameters evaluated were: body weight, abdominal circumference, volume of ascitic fluid and tumor cells, viable and nonviable tumor cell count, determination of mean survival time and increased life-span. Both extracts presented antitumor activity, but SFE reduced more the volumes of ascites fluid and the tumor cells (4±1 and 1±0.4mL, respectively), while caused higher mean survival time (17 days) and increased life span (∼31%). The results suggest the importance of the polyacetylenes from B. pilosa as leader molecules to contribute to a new anticancer drug by using the supercritical technology.