Co-encapsulated doxorubicin (DOX) and curcumin (CUR) in poly(butyl cyanoacrylate) nanoparticles (PBCA-NPs) were prepared with emulsion polymerization and interfacial polymerization. The mean particle size and mean zeta potential of CUR-DOX-PBCA-NPs were 133 ± 5.34 nm in diameter and +32.23 ± 4.56 mV, respectively. The entrapment efficiencies of doxorubicin and curcumin were 49.98 ± 3.32% and 94.52 ± 3.14%, respectively. Anticancer activities and reversal efficacy of the formulations and various combination approaches were assessed using 3-[4,5-dimethylthiazol-2-yl] 2,5-diphenyltetrazolium bromide assay and western blotting. The results showed that the dual-agent loaded PBCA-NPs system had the similar cytotoxicity to co-administration of two single-agent loaded PBCA-NPs (DOX-PBCA-NPs+CUR-PBCA-NPs), which was slightly higher than that of the free drug combination (DOX+CUR) and one free drug/another agent loaded PBCA-NPs combination (DOX+CUR-PBCA-NPs or CUR+DOX-PBCA-NPs). The simultaneous administration of doxorubicin and curcumin achieved the highest reversal efficacy and down-regulation of P-glycoprotein in MCF-7/ADR cell lines, an MCF-7 breast carcer cell line resistant to adriamycin. Multidrug resistance can be enhanced by combination delivery of encapsulated cytotoxic drugs and reversal agents.