P-glycoprotein (P-gp, ABCB1) is an important part of the multixenobiotic resistance (MXR) defense system in aquatic organisms. The main goal of this study was identification of P-gp inhibitors in contaminated sediments using the effect-directed analysis (EDA) approach. The samples were collected from the Gorjak creek (Zagreb, Croatia), a recipient of wastewater effluents from the pharmaceutical industry. Sediment samples were extracted and fractionated using two-tiered approach. Resulting non-polar, medium polar and polar fractions were tested on the inhibition of P-gp activity using P-gp overexpressing PLHC-1/dox cells and calcein-AM as model substrate. The obtained EC50 values - up to 757 μg/g, expressed in toxicity equivalents of model P-gp inhibitor cyclosporine A - revealed high inhibitory potential of polar fractions of investigated sediments and clearly reflected the impact of pharmaceutical wastewater. P-gp specific ATPase assay and the cytotoxicity modulation experiments with colchicine indicated that most of the observed P-gp inhibition was due to the presence of non-competitive inhibitors. A detailed chemical analysis by ultrahigh-performance liquid chromatography-quadrupole/time-of-flight mass spectrometry (UPLC-QToFMS) revealed nonionic surfactants, including alcohol polyethoxylates (LAEOs) and polypropylene glycols (PPGs), as the major components of the most active sub-fractions. Testing of several LAEO and PPG commercial mixtures confirmed their potential to inhibit the fish P-glycoprotein and modulate toxicity of other xenobiotics present in complex environmental samples.