This study evaluated the antioxidant activity and total phenolic content (TPC) of Butia odorata fruit extracted with different solvents, as well as the phenolic composition using UHPLC-MS/MS analysis. In addition, the antitumor potential of B. odorata fruit against various cancer cell lines (Caco-2, HeLa, SiHa, C33a) was evaluated for the first time. The TPC and antioxidant activity (DPPH• and ORAC assays) varied according to the extraction solvents, with the highest values obtained for the methanol:water (80:20, v/v) extracts. Thirteen phenolic compounds were quantified in the ethanol:water (80:20, v/v) extracts of B. odorata fruit by UHPLC-MS/MS, with the main compounds identified as (+)-catechin (259 ± 18 mg kg−1), (−)-epicatechin (211 ± 12 mg kg−1) and rutin (161 ± 2 mg kg−1). Sinapic and ellagic acids, trans-resveratrol, naringenin, and apigenin were reported in B. odarata fruit for the first time. Furthermore, the B. odorata fruit showed antitumor activity against cervical cancer cell lines (SiHa and C33a), providing preliminary evidence for further assessment of its anticancer potential.