The inhibitory effect of flavonoids on the metabolic activity of the cytochrome P450 enzyme was tested using the biotransformation reactions: 6β-hydroxylation of testosterone (CYP3A4), O-demethylation of dextromethorphan (CYP2D6), O-deethylation of phenacetin (CYP1A2), coumarin 7-hydroxylation (CYP2A6) and 6α-hydroxylation of paclitaxel (CYP2C8). The residual activity of cytochrome P450 enzymes was determined using HPLC-DAD analytical method. Seven flavonoids showed statistically significant potential for CYP3A4 inhibition (P < 0.05), five flavonoids showed CYP1A2 inhibition, while the results of the CYP2A6 enzyme inhibition assay showed significant inhibitory potential of four flavonoids. None of the flavonoids tested showed statistically significant CYP2D6 inhibition. Three flavonoids, inhibited CYP2C8 enzyme activity. For the most potent inhibitors (acacetin, chrysin, apigenin), the most likely conformation in the active site of the CYP3A4, as well as the potential binding site, was determined by molecular docking study. Docking results showed that the binding energies of the molecular spices in the CYP3A4 active site are lower than the anion spices. All the top-ranked molecular species are oriented with ring B toward heme iron. Quantitative structure activity relationship (QSAR) analysis was performed using the random forest method and the hierarchical clustering method. All selected descriptors indicate that a significant hydrophobic B ring, interacting with the heme iron, is required for significant inhibition of CYP3A4. The hierarchical cluster formation showed that tested flavonoids equally successfully inhibit CYP3A4, CYP1A2, CYP2A6 and CYP2C8. In the case of CYP2A6, a statistically significant dependence of residual activity and the highest occupied molecular orbital energy (EHOMO) was further established.