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Background: The successful launch of icaritin, a therapeutic drug for liver cancer derived from Epimedium brevicornu, has provided new impetus for the development of prenylated flavonoids in the field of oncology. Flemingia macrophylla is reported to contain characteristic prenylated flavonoids which can regulate the p53 protein. We aimed to isolate these constituents and conduct activity evaluation, structure–activity relationship, and mechanism studies to provide candidate compounds for antitumor drug development. Methods: In this study, chromatographic techniques combined with spectroscopic methods were used to separate, purify, and identify the constituents of Flemingia macrophylla methanol extract. The cytotoxic activity of the constituents was evaluated using an MTT assay with A549 and H1975 cells as the model. The binding mechanism between the compounds and the p53 protein was investigated with molecular docking and validated with cellular thermal shift assay (CETSA). Western blotting (WB) was employed to detect the expression of p53 protein and apoptosis-related proteins in cells. Results: Chiral HPLC separation of racemates 1 and 7 provided two pairs of undescribed enantiomers (1a/1b and 7a/7b), along with eight known compounds (2 − 9) isolated from Flemingia macrophylla roots. Their structures were elucidated by spectroscopic analysis, and the absolute configurations of the enantiomers were determined from experimental and calculated electronic circular dichroism data. Compounds 1 − 7, and the non-prenyl analogues 10 − 13, were evaluated for cytotoxic activity against the human lung cancer A549 and H1975 cell line. Compounds 5 − 7 displayed better cytotoxicity than the positive control icaritin in A549 and H1975, with IC50 values ranging from 4.50 to 19.83 μmol·L-1 and < 5 μmol·L-1, respectively. The structure–activity relationships of the chromone or flavonoid analogues against A549 cells were discussed. Molecular docking results demonstrated that compound 7a has strong interaction with p53 and WB indicated that 7a induced apoptosis by increasing the p53 protein, decreasing the anti-apoptotic protein Bcl-2, and activating the caspase family in A549 cells. These results suggest that prenylated flavonoids are potential p53 protein activators. Conclusion: This study demonstrates that Flemingia macrophylla is rich in prenylated flavonoid constituents, among which compounds 5 and 7 exhibited significant cytotoxic activity against A549 cells and served as reference candidates for the design and development of prenylated compounds as antitumor therapeutic drugs. Highlights: Two pairs of undescribed enantiomers (1a/1b and 7a/7b) were isolated from Flemingia macrophylla. Prenylated flavonoids 5 − 7 exhibited significant cytotoxicity against A549 cells. Structure-activity relationship study revealed that the presence of B-ring and 2ʹ-OH substitution and the cyclization of 6/8-pentenyl group with 7-OH were beneficial for cytotoxicity. Molecular docking results showed that Cys124 and Cys277 were the key amino acid residues for binding. Compound 7a induced apoptosis of A549 cells by activating the p53 protein. [ABSTRACT FROM AUTHOR]