The flower of Pueraria lobata (Puerariae Flos) is a reddish-purple to violet-purple flower that blooms between July and September. In our preliminary study, Puerariae Flos extract exhibited significant activity against a human ovarian cancer cell line. This research aims to identify the active compounds in Pueraria Flos. By repeated chromatography, one new tryptophan derivative (1), two new flavanones (4 and 5), and 19 known compounds, including tryptophan derivatives (2 and 3), flavonoids (6-9), isoflavonoids (10-20), a flavonolignan (21), and a phenolic compound (22), were isolated from a methanol extract of Puerariae Flos. The structures of new compounds were elucidated as 13-N-benzoyl-l-tryptophan-1-N-β-d-glucopyranoside (1), 2-hydroxy-5-methoxy-naringenin (4), and 2-hydroxy-5-methoxy-naringenin 7-O-β-d-glucopyranoside (5). Among the isolates, afromosin (17), tectorigenin (11), apigenin (8), glycitein (16), (-)-hydnocarpin (21), irilin D (12), irisolidone 7-O-glucoside (14), and genistein (10) showed cytotoxicity against human ovarian cancer cell line A2780. Apigenin (8) and (-)-hydnocarpin (21) were the most active (IC50 values of 9.99 and 7.36 μM, respectively).