Two biotinylated derivatives of the main hop chalcone xanthohumol (1) were prepared by a one-step synthesis via esterification using biotin and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC HCl) and 4-dimethylaminopyridine (DMAP) as coupling reagents. The products were characterized spectroscopically and their antiproliferative activity toward MCF-7, MCF-10A, HepG2, MDA-MB-231, 4T1 and Balb/3T3 cell lines was investigated using the SRB assay. For all three tested compounds the best activity was noted in the case of human (MCF-7) and mice (4T1) breast cancer cell lines (IC₅₀ values < 9 μM). Both biotinylated derivatives showed higher anticancer activity than xanthohumol (1) towards all types of tested breast cancer cells. Double biotinylated xanthohumol (3) proved to be the most active in inhibiting cell growth, with IC₅₀ values equal to 5.35 ± 1.5 μM for 4T1 and 8.03 ± 0.53 µM for MCF-7 cell lines. Compound 3 was also more active than 1 and 2 against liver cancer cells HepG2 (IC₅₀ = 17.37 ± 5.1 μM), while the IC₅₀ values for 1 and 2 were equal to 21.5 ± 2.7 and 22.1 ± 3.9 µM, respectively. 4‑O‑biotinylxanthohumol (2) was the second most active growth inhibitor, particularly with respect to MCF-7 (IC₅₀ = 6.19 ± 1.7 μM) and 4T1 (IC₅₀ = 6.64 ± 0.4 μM) cell lines. The antioxidant activity was evaluated using the 1.1-diphenyl-2-picrylhydrazyl radical (DPPH) method. All tested compounds (1-3) have antioxidant activity between 2.73 and 3.38 mM. It was reported for the first time that new prenylated chalcones containing the biotin moiety effectively inhibited proliferation of cancer cells in vitro.