Plants on this planet are essential need to support life, its needs of acquired ecosystem yet many of the fears challenges including the conservation, especially their germplasm and same is being feared in case of walnut in Kashmir, Pakistan. Walnut (Juglans regia L.) is the exceptionally widespread nut tree in the world and grow best in northern regions of Pakistan and Azad Jammu and Kashmir. It has wide range of genetic diversity which indicate that interspecific hybrids exist and introgression may occur. Plants on this planet are coupled with continuous challenges and their germplasm conservation is one of the prime priority. We focused to evaluate the genetic characterization of sixty four walnut accessions including their genetic relationship and distance collected from sixteen selected sites of Neelum valley Azad Jammu and Kashmir in Himalayan region of Pakistan by using thirty five primer combinations during SSR analysis. Genetic distance among these accessions were displayed in a dendrogram cluster analysis correlation based and principal component analysis (PCA). We found that six cluster were clearly separating the accessions from each other and distance among accessions confirmed that these accessions were divergent due to geographical diversity, naturalized population, unevenness and interspecific cross pollination. The close relationship among accessions showed these accessions had probably same ancestory. Moreover, fieldwork study revealed that local walnut germplasm are being eroded from some regions due to urbanization, agriculture, grazing, wood harvesting and natural calamities (earth quake and land sliding) that depicted genetic drift. In the actual application, the measurement of genetic diversity among these accessions should be used as important guidelines for the researchers, conservators, plant breeders and regulatory authorities while ensuring that a substantial genetic diversity is preserved within the study area. Medicinal plants have a proven long history for their effective use to prevent and treat diseases. During the recent decades, the modern drug discovery approaches have refocused on traditional medicinal plants because of their higher contribution for providing new drugs, drug candidates, novelty and mechanism insights. Juglans regia is very famous for providing precious and medicinal dry fruits which are extensively being used in many parts of the world. Walnut is a rich and diverse source of natural products, including variety of phenolic and flavonoid natural products. These natural products possess variety of biological properties, especially anti-oxidant and anti-microbial activities. Anti-oxidant reduce free radicals by their lone-pair of electron and decrease the level of free radicals in body. Anti-microbial activities in order to cure ailments, discovery of new drugs is a prime global priority and going back to natural treasure of medicinal plants is one of the best option. Human challenges for the search of new drugs are also burdened with the current status of diseases caused by oxidative stress as well as the limitation of existing antioxidant drugs. Toxic metal contaminations are the critical issue especially in dietary and medicinal plants like J. regia because these metals unwantedly modulate various biological processes which results into various diseases for example, they damage tissue, organ and results into various diseases including cancer, ulcer and neurological disorders. In this study we used four parts of J. regia i.e. fruits, leaves, stem bark and green husk. Metal contamination estimation, using atomic absorption spectroscopy, found that Cd contamination in its fruits, Cu and Mg concentration in all parts of J. regia (fruits, leaves, stem bark and green husk) are critically above the standard permissible limits and thus is a serious health concern. Methanolic extracts of various parts of J. regia were screened for antimicrobial activities against twelve different human pathogens all extracts indicated promising antimicrobial activities in comparison to the standard clinical drugs, ciprofloxacin and nystatin, among all the tested plant parts maximum zone of inhibition were measured in fruits extract (14.33 ± 0.33) against S. aureus, leaves (19.33 ± 0.33) against E. coli , stem bark (16.33 ± 0.57) against B. subtilis and green husk (18.16 ± 0.57) against P. aeruginosa of J. regia extracts Whereas, antioxidant studies on J. regia revealed that its fruits has the highest antioxidant properties with IC50 value of 6.938693 mg/mL. Total phenolic contents were estimated spectrophotometrically with Folin-Ciocalteau reagent and were calculated as gallic acid (GA), (equivalents mg of GA/g of extract which ranged from 12.6987 to 31.9685 mg GA/g). Whereas, total flavonoid contents of J. regia were estimated as rutin equivalents (mg of Rutin/g of extract) and highest flavonoid contents were found in the fruit’s extract which was 25.8231 mg/g and lowest in leaves extract of J. regia 7.2861 mg/g. Cancer is a devastating disease and one of the leading causes of death worldwide despite improvements in patient survival during the past 50 years; new and improved treatments for cancer are therefore actively sought. Breast cancer is the top most prevalent cancer in women worldwide and is responsible for nearly 58 % of all cancer deaths. Our search for anti-cancer medicinal plant Juglans regia fruits, green husk, and leaves showed promising antiproliferative activities in MCF-7 breast cell lines, whereas stem bark showed moderate activity, hightest recorded among all extracts, fruit of J. regia showed highest antiproliferative activity with GI50 1.581722. Advancement in human knowledge at molecular and atomic level initiated researches for the search of individual molecules responsible for specific biological properties. This together with the development in equipment gave birth to molecular medicine in late 19th and early 20th century. Importantly, medicinal plants have been the superlative precursor for the discovery and development of molecular pharmaceuticals. Therefore, we designed and conducted isolation of pure natural products from Juglans regia and elucidate their structures using modern chromatography and spectroscopic techniques as a minor component of this research. We isolated 23 natural products and were able to elucidate the structures of three of them which were found to be known juglone, eugenol and 5-Hydroxy-3, 7, 4-trimethoxyflavone.