The current investigation was carried out to develop a sustainable dyeing method by utilization of a marine brown seaweed Cystoseira indica as natural dye. Moreover, the dyeing process was optimized by varying fixing agent percentage and dyeing time. The influence of different variables on color uptake (K/S), fastness rating and color measurement were examined. The bioactive efficiency of seaweed extract and dyed fabric were also investigated against human pathogens such as Escherichia coli, Staphylococcus aureus and Candida albicans and were compared with commercial antibacterial and antifungal drugs. It was found at the optimized dyeing condition has 1 percent formic acid as fixing agent with 120 min dyeing period. At this condition, the dye molecule shows 70 percent exhaustion and 59 percent fixation along with acceptable fastness and antimicrobial activities Furthermore, the dyed fabric shows effective inhibition against all tested organisms. Fabric samples showed greater antimicrobial efficiency for Candida albicans and poorer than for Escherichia coli.