Background: Black cumin oil is obtained from the seeds of Nigella sativa L. which belongs to family Ranunculaceae. The seed oil has been reported to possess antitumor, antioxidant, antibacterial, anti-inflammatory, hypoglycemic, central nervous system depressant, antioxidant, and immunostimulatory activities. These bioactivities have been attributed to the fixed oil, volatile oil, or their components. Seed oil consisted of 15 saturated fatty acids (17%) and 17 unsaturated fatty acids (82.9%). Long chain fatty acids and medium chain fatty acids have been reported to increase oral bioavailability of peptides, antibiotics, and other important therapeutic agents. In earlier studies, permeation enhancement and bioenhancement of drugs has been done with black cumin oil. Objective: In order to recognize the mechanism of binding of fatty acids to P-glycoprotein (P-gp), linoleic acid, oleic acid, margaric acid, cis-11, 14-eicosadienoic acid, and stearic acid were selected for in silico studies, which were carried out using AutoDock 4.2, based on the Lamarckian genetic algorithm principle. Materials and Methods: Template search with BLAST and HHblits has been performed against the SWISS-MODEL template library. The target sequence was searched with BLAST against the primary amino acid sequence of P-gp from Rattus norvegicus. Results: The amount of energy needed by linoleic acid, oleic acid, eicosadienoic acid, margaric acid, and stearic acid to bind with P-gp were found to be − 10.60, −10.48, −9.95, −11.92, and − 10.37 kcal/mol, respectively. The obtained data support that all the selected fatty acids have contributed to inhibit P-gp activity thereby enhances the bioavailability of drugs. Conclusion: This study plays a significant role in finding hot spots in P-gp and may offer the further scope of designing potent and specific inhibitors of P-gp.