Medicinal plant-based dyeing introduces vibrant colours alongside antibacterial and anti-odor properties, fostering sustainability. Innovations include mass sampling for consistent pigment production. Furthermore, lotus seedpods, rich in bioactive compounds, exhibit potential in diverse fields. Chitosan, a biopolymer derived from chitin, boasts versatility in drug delivery systems, wound healing, and antimicrobial applications. This experimental inquiry delves into the dyeability and antimicrobial efficacy of cotton fabric subjected to treatment with chitosan and dyed using Lotus seeds. The investigation encompasses varied parameters such as dye concentration, duration, and temperature for fabric dyeing. The study elucidates the aqueous extraction methodology for dye from Lotus seeds, the chitosan-based mordanting techniques. Standardized assessments are utilized to gauge colour fastness characteristics, encompassing washing, rubbing, and perspiration fastness, along with assessing the antimicrobial attributes of the treated cotton fabric. The study indicates that the application of chitosan-treated cotton fabric enhances dye intensity and overall dyeing quality, consequently improving the colour fastness property of washing, rubbing, and perspiration. Additionally, the treated cotton fabric exhibits significant antimicrobial activity against common bacteria, highlighting the potential of Lotus seed-derived colourants and chitosan mordant in the development of sustainable and antimicrobial textiles. The future scope lies in optimizing the integration of Lotus seed-derived colourants and chitosan mordant for enhanced sustainability and antimicrobial efficacy in textile applications, leveraging advancements in dyeing technology and antimicrobial formulation techniques.