The ubiquitous presence of pharmaceuticals in aquatic systems is a challenging problem as their potential chronic effects on aquatic organisms remain largely unknown. The ATP-binding cassette (ABC) transport proteins contributing to the multidrug/multixenobiotic resistance (MDR/MXR) phenomenon seem to have an important role in the elimination of xenobiotics in aquatic organisms. Modulation of their efflux activities by contaminants may lead to substantial increase in intracellular accumulation and toxic effects of other xenobiotics. The aim of our work was to analyse a series of pharmaceuticals for their potential to modulate the activity of xenobiotic efflux transporters from the ABCB and ABCC sub-family in the Poeciliopsis lucida hepatoma cell (PLHC-1) fish cell line (PLHC-1/wt) and a doxorubicin (DOX) resistant PLHC-1 subclone (PLHC-1/dox) characterized by an elevated expression of the P-glycoprotein (ABCB1). Cellular accumulation of the model fluorescent substrates calcein-AM and rhodamine123 were used to determine an inhibitory effect on P-gp1 and/or MRP-like efflux transporters. 18 out of 33 tested pharmaceuticals showed MXR inhibitory activity with IC50 values occurring in the lower micromolar to millimolar range. Further, cytotoxic effects of pharmaceuticals were evaluated in PLHC-1/dox cells. Co-exposure of resistant cells to model P-gp1 inhibitor cyclosporine A (CyA) resulted in up to five times increased cytotoxicity of pharmaceuticals. In addition, some pharmaceuticals lead to a marked increase in cytotoxicity of doxorubicin, a model P-gp1 substrate. The modulation of toxicity by MDR inhibitors indicates their role in influencing cellular toxicity. In conclusion, the results of our study revealed significant inhibitory effects of environmentally relevant pharmaceuticals on P-gp1 and MRP-like transporters in fish. Our findings correspond well with data from mammalian systems indicating that the specificity and roles of the related efflux transporters may be similar in fish. Furthermore, due to the presence of active and inducible ABC transport proteins, the PLHC-1 cells appear to be a reliable in vitro system for the investigation of MDR/MXR mechanisms in fish.