Background: Mitochondrial dysfunction is the major cause of various types of cancer, leading to death worldwide. The present study investigated the in silico binding potential of natural flavonoids and essential oils with human cyclophilin D (CyPD) protein. CyPD protein is a major molecular marker for apoptosis and has been reported to be elevated in oral carcinoma. Methods: PubChem database was used to check the efficacy of different active phytoconstituents (kaempferol, quercetin, eugenol, oxyresveratrol, tanshinone 2a, catechin, epicatechin, cinnamaldehyde, and emodin). These compounds were used as ligands to check their potential as anticancer agents against the inner mitochondrial membrane protein, CyPD. Docking studies were performed with the help of Discovery Studio 2.5 and Autodock. Emodin was used as a reference inhibitor to compare the results. Results: The binding energy (B.E.) of the reference inhibitor (known/established drug) emodin was observed −28.9 kcal/mol while novel inhibitors (catechin, cinnamaldehyde, epicatechin, eugenol, kaempferol, oxyresveratrol, quercetin, and tanshinone 2a) exhibited a range from −51.51 to −5.89 kcal/mol. Quercetin, kaempferol, and epicatechin (B.E.: −51.51, −34.79, and −30.62 kcal/mol, respectively) showed strong affinity as compared to reference inhibitor (B.E.: −28.9 kcal/mol). Conclusion: Quercetin, kaempferol, and epicatechin can be used as lead inhibitors against targeting CyPD.