Edifenphos (EDF), an important organophosphate fungicide used in agriculture, is a great threat to human health and environment. To assess the toxicity of EDF at the level of protein molecule, the effect of EDF on human serum albumin (HSA) was investigated by biophysical and biochemical approaches. EDF-HSA complex is formed as a result of static quenching as revealed by the intrinsic fluorescence analysis. Thermodynamic analysis of the binding data suggests involvement of hydrophobic interactions in EDF-HSA complex formation, which is in line with molecular docking results. Moreover, thermodynamic parameters of binding between EDF and HSA suggest entropy-driven spontaneous interaction, presumably dominated by hydrophobic forces. Further, binding site of EDF seems to have been located within sub-domain IIA of HSA. EDF binding to HSA decreases its alpha helical content as analyzed by CD spectra. Marked micro-environmental changes around tryptophan/tyrosine residues in HSA upon EDF binding were recorded via three-dimensional fluorescence spectroscopy. Substantial release of protein carbonyl from HSA as a result of EDF treatment suggested involvement of ROS in EDF induced protein damage. This work is expected to provide some leads toward EDF induced toxicity in humans and would be helpful in reinforcing the check on food safety.