Since the last decade, the application of natural dyes on textile material has been gaining popularity all over the world, possibly because of the increasing awareness of issues concerning the environment, ecology and pollution control. The present paper investigates extraction of natural dye from leaves of the plant Vitex negundo, which is an abundant, cheap, and readily available agricultural by-product. Water extracts from V. negundo was used to dye silk fabrics. Optimum extraction conditions included pH 9, duration 120 min, and temperature 90°C. Optimum dyeing conditions included dyeing pH 5 and duration of 60 min. Potash alum, tannic and tartaric acid were used as mordants, all of which are benign to human health and the environment. Color strength and color coordinates in terms of L*, a*, b*, C, and h were examined. A range of shades were obtained when fabrics were dyed with different mordants and mordanting techniques. The extracted dye was tested for some of the eco-parameters using atomic absorption spectrophotometry and GC/MS. The test results were compared with set standards to determine the eco-friendliness of natural dye. Their concentrations were found to be lower than the stipulated limits. Dyed samples were tested for antimicrobial activity against gram-positive and gram-negative bacteria. The dyed silk fabrics showed acceptable fastness properties and were also found to possess antibacterial activity. It can be concluded that the abundantly available agricultural byproduct V. negundo has great potential to be effectively utilized as a natural dye for silk.