The hexanic and ethanolic extracts obtained from Rollinia laurifolia, collected in Minas Gerais State/Brazil, were screened by the brine shrimp lethality and cytotoxic assays on different cell lines, in order to identify potential sources for novel antitumoral compounds. Fifty percent of the ethanolic derived fractions (E) showed significant cytotoxic activity (IC₅₀ < 10–1 μg/mL), not related to tannins or saponins, i. e., IC₅₀ E13 on C6 = 1.54 x 10^-2 μg/mL, IC₅₀ E29 on L₉₂₉ and C₆ = 6.43 x 10^-1 μg/mL and 3.57 x 10^-2 μg/mL, respectively. RP-HPLC analysis led to the purification and identification of two known acetogenins, gonionenin and annonin-I, detected for the first time in Rollinia sp, and a new acetogenin, named laurifolin.