Three new series of chromene molecules have been synthesized in order to explore their antimicrobial activity. The series encompass 2-substituted 14-(4-halophenyl)-12-methoxy

β-Enaminonitriles bearing 9-hydroxy-1H-benzo[f]chromene moiety was synthesized. The targeted compounds were evaluated for their anti-proliferative activity against three human tumor cell lines, PC-3, SKOV-3 and HeLa, and the active cytotoxic compounds were further evaluated against cancer cells, MCF-7/ADR, and two normal cell lines, HFL-1 and WI-38. Few compounds were assigned to be the most potent derivatives against PC-3, SKOV-3 and HeLa cell lines in comparison with Vinblastine and Doxorubicin. Several compounds possessed a relatively good potency against MCF-7/ADR cells as compared with Doxorubicin and were tested as a P-gp inhibitor. Moreover, the halogenated substituents, 2,4-F2, 2,3-Cl2, 2,5-Cl2 and 3,4-Cl2; have good potency against P-gp-mediated MDR in MCF-7/ADR as compared with Doxorubicin. Meanwhile, Rho123 accumulation assays revealed that few compounds effectively inhibited P-pg and efflux function. In addition, certain derivatives induced apoptosis and an accumulation of the treated MCF-7/ADR cells in the G1, S and G1/S phases.