In this study, the efficacy of water lily(Nymphaea tetragona) leaves as a new natural dye resource was investigated. For this purpose, the colorants from water lily leaves were extracted in methanol, evaporated, and powdered. Dyeing onto cotton fiber was carried out to study the effects of dyeing conditions, mordant type and mordanting method on dye uptake, color change, and colorfastness. FTIR analysis supported that hydrolyzable tannins and chrolophyll were contained in the extracted colorants. The colorants showed good affinity to cotton fiber showing Y Munsell color. Pre-mordanting method gave better results in terms of dye uptake than post-mordanting method. By mordanting, dyed fabrics exhibited various colors such as green, khaki, brownish yellow, dark brown, dark gray and so on. Colorfastness to washing and rubbing was relatively good showing 4-5 rating. The light fastness was improved 1-2 rating by Fe mordanting. The dyed cotton fabric showed antimicrobial activity.