Dyeing of cotton, viscose, wool, and polyester/viscose blended fabrics has been carried out with the aqueous extract of the roots of Alkanna orientalis plant. Fastness properties and color strength values were investigated in terms of users. Phenolic compounds and cytotoxic activities of ethanol (EtOH) and water extracts were determined. It was found that cotton fabric dyed with the aqueous extract of the roots of A. orientalis plant in the presence of alum mordant gave better depth of shade (K/S). The toxicity studies reveal that the EtOH and the water extracts of the root of A. orientalis exhibited 0% cytotoxicity while the standard cancer drug 5-fluorouracil exhibited 6% cytotoxicity. The overall results of this study show that the roots of A. orientalis can be used in the textile industry, especially for dyeing cotton fabrics.