Introduction: Myrica nagi Thunb. (family Myricaceae) are actinorhizal plants showing symbiotic interaction with Frankia. Inhibition of cyclooxygenase-2 (COX-2) enzyme is known to be significant in preventing inflammation and in therapeutics. Objectives: Our principal focus was to identify COX-2 enzyme inhibitors, safer and natural anti-inflammatory compounds from M. nagi. Protein–ligand interaction has a significant role in structure-based drug design. Materials and Methods: Sixty-eight phytochemicals were therefore screened and evaluated for their binding energies with COX-2. These phytoconstituents were screened and analyzed for drug Likeliness along with Lipinski's rule of five. The X-ray crystallographic structure of the target COX-2 (protein data bank [PDB] ID: 4PH9), obtained from PDB, was docked with PubChem structures of phytochemicals using AutoDock 4.2 that uses Lamarckian genetic algorithm. Further, myricetin was subjected to in vitro anti-inflammatory assay using RAW-264.7 cell lines and inhibitory concentration (IC50) value was also determined. Results: The myricetin, myricitrin, and corchoionoside-C inhibited COX-2 with − 6.52, −4.94, and − 4.94 Kcal/mol binding energies, respectively, comparable to ibuprofen. Eventually, bioactivity score and absorption distribution metabolism excretion-toxicity properties showed considerable biological activities as G protein-coupled receptor, nuclear receptor, protease inhibitor, and enzyme inhibitors for myricetin, myricitrin, and corchoionoside-C phytochemicals. Molecular docking revealed hydrophobic interactions followed by four, nine, and four numbers of hydrogen bonds between myricetin, myricitrin, and corchoionoside-C, respectively, within the binding site of COX-2. Flavonol myricetin showed 112 μg/mL as IC50 value when it was subjected to in vitro cytotoxicity assay. These results clearly demonstrated that myricetin, myricitrin, and corchoionoside-C could act as highly potential COX-2 inhibitors. Therefore, in silico and in vitro studies revealed that of three best phytochemicals, myricetin could be promising candidate.