DNA barcoding is been proposed as a powerful taxonomic tool for species identification genetically. The Consortium for the Barcode of Life (CBOL) Plant Working Group has recommended the combination of rbcL & matK as the core plant barcode. Rhododendron of Nepal shows genetic diversity due to which 31 different species were reported till date, found in Nepal based on morphological character. We collected different samples from the central and western parts of Nepal (Province 3 & 5). For the phytochemical study, cold extraction was performed using methanol as solvents. The crude extracts were tested for the presence of phytochemicals such as alkaloids, saponins, glycosides, tannins, flavonoids, and coumarins. The same extracts were used for antimicrobial assay five different pathogenic micro-organisms. MIC was done for calculating the minimum effective concentration of the extract. Then TLC was done to separate the compound. The methanol extract was analyzed for core secondary metabolites by HPLC-QTOF and found more than 20 medicinal important compounds. DPPH assay was also done with crude extracts for identifying antioxidant potency of the extracts. Among samples compared, Gulmi and Palpa showed the best result for antioxidant assay with minimum IC50. Similarly, phenolic content was also checked and samples from Palpa showed high phenolic content. The rbcL and MatK gene loci show the 99.98% similarly for the R.arboreum in NCBI database and were submitted to the BOLD database for the final authentication and get success for the first time to get published DNA barcode of Nepalese plants.