Extrusion of drugs or drug-like compounds through bacterial efflux pumps is a serious health issue that leads to loss in drug efficacy. Combinatorial therapies of low-efficacy drugs with efflux pump inhibitors may help to restore the activities of such drugs. In this quest, natural products are attractive molecules, since in addition to their wide range of bioactivities they may inhibit efflux pumps. The current work repurposed the bioactive alkaloid roemerine as a potential efflux pump inhibitor. In Bacillus subtilis, both Bmr and BmrA, belonging to the major facilitator and the ATP-binding cassette superfamilies, respectively, were found to be inhibited by roemerine. Scanning electron microscopy and RNA-Seq analyses showed that it potentiated the effect of berberine. Growth rates and checkerboard assays confirmed the synergy of roemerine and berberine and that roemerine prevented berberine efflux by inhibiting Bmr. Transport assays with inverted membrane vesicles prepared from Escherichia cob overexpressing BmrA showed that increasing roemerine concentration decreased the transport of doxorubicin, the BmrA substrate, confirming that roemerine may also be considered as an inhibitor of BmrA. Thus, these findings suggest that conjugation of roemerine to substrates of efflux pumps, Bmr and BmrA, may help to potentiate the activity of their drug substrates.