Researchers are continuously focusing and exploring new aspirants in the field of natural dyeing to reduce the application of synthetic dyes and their harmful effect on living organisms via natural dyes. This aimed the evaluation on dyeing ability of a novel natural dye extracted from root bark of C. polygonoides, their pharmacological significance and phytotoxicity. Biological analyses of methanolic dye extract were conducted in laboratory according to standard protocols. Phytochemicals analysis of the dye solution indicated that it was rich in natural phenolics. Antibacterial activities were carried out in-vitro against various strains of bacteria such as Klebsiella pneumoniea, Staphylococcus aureus, Acinetobacter baumannii and Escherichia Coli. Maximum zone of inhibition against E. Coli (13 mm), K. pneumoniea (20 mm), A. baumannii (17 mm) and S. aureus (15 mm) was recorded when dye was used at 3mg/ml in dimethyl sulfoxide (DMSO). For antifungal activity, the dye at 3 mg/ml in DMSO was highly effective against A. niger showing 17 % inhibition in fungal growth and A. flavous showing 4% inhibition in linear growth. We used DPPH radical scavenging assay to document the antioxidant potential of the dye extract. The maximum scavenging potential of dye extract was found at 400 μg/ml (48.3%±0.3). Phytotoxic effect of C. polygonoides dye was conducted by the germination of maize in various concentrations (250-1000 ppm). The dye did not show any phytotoxic effect on the development and growth maize seedlings. We also checked the dye for its dying potential on cotton fibers. It was observed that the dye stained the cotton fabric as bright red using acetic acid as a mordant. Moreover, the dye was resistant to continuous washing with tap water and after that dye was considered as not washable. We concluded from these studies that dye extracted from root bark of C. polygonoides is antibacterial, shows antioxidant properties, non-toxic and efficient in dyeing of cotton fibers.