Glutathione transferases (GSTs) are a family of Phase II detoxification enzymes that are involved in the development of multi-drug resistance (MDR) phenomena toward chemotherapeutic agents. GST inhibitors are considered candidate compounds able to chemomodulate and reverse MDR. The natural flavonoid myricetin (MYR) has been shown to exhibit a wide range of pharmacological functions, including antitumor activity. In the present work, the interaction of MYR with human glutathione transferase A1–1 (hGSTA1–1) was investigated by kinetics inhibition analysis and molecular modeling studies. The results showed that MYR binds with high affinity to hGSTA1–1 (IC50 2.1 ± 0.2 μΜ). It functions as a non-competitive inhibitor towards the electrophile substrate 1-chloro−2,4-dinitrobenzene (CDNB) and as a competitive inhibitor towards glutathione (GSH). Chemical modification studies with the irreversible inhibitor phenethyl isothiocyanate (PEITC), in combination with in silico molecular docking studies allowed the prediction of the MYR binding site. MYR appears to bind at a distinct location, partially overlapping the GSH binding site (G-site). The results of the present study show that MYR is a potent inhibitor of hGSTA1–1 that can be further exploited towards the development of natural, safe, and effective GST-targeted cancer chemosensitizers.