Dyeing also known as coloration is an important process of imparting color to products and provide the desired aesthetic appeal, fashion, and function thus increase attractiveness and marketability. Synthetic dyes used for dyeing purpose in today’s world are not only based on non-sustainable petrochemical oils but also their production and use has become a cause of concern for environmental pollution and health problems. In this respect, the demand for eco-friendly natural dyes as coloring agents is continuously increasing. With these points, this study was conducted to produce natural dye and evaluate its cotton fabric dyeing properties. Natural dye was extracted by maceration extraction method using 70% methanol as a solvent. Total flavonoids and phenol were quantified by spectrophotometric method. By the preliminary work, Agave sisalana was selected from (Rumex Nervousus Vahl flower, Syzygium Guineensis, and Carissa Edulis fruit peel) as natural dye source for dyeing cotton fabric. Because Agave sisalana natural dye contains highest total flavonoids (7.52 ±0.255 mg QUE/g DW) and the color strength of cotton fabrics dyed by Agave sisalana natural was highest (6.645) in the case of cotton fabric mordanted by FeSO4 compared to other plant natural dyes. Phytochemical analysis as well as UV/VIS and Fourier transform infrared spectroscopy shows flavonoids, anthocyanins, and tannins were present in Agave sisalana natural dye while anthraquinone and alkaloids were not present. The physicochemical properties such as moisture content (4.89 ±0.274%), water activity (0.56 ±0.011), and hygroscopicity (28.94 ±0.97 g/100g) of Agave sisalana natural dye were found to be below the tolerance limit of (moisture 5% and αw ˂0 .60) for spoilage. Agave sisalana natural dye was highly soluble in 50% methanol (89.66 ±2.084%) and hot water (83.41 ±1.745%) than in hexane (52.084 ±2.251%) and cold water (68.37 ±1.232%). Dyeing time, dye concentration, and temperature significantly affects the adsorption of dye onto cotton fabrics and the color strength of dyed fabrics. The optimum (maximum) values of dye adsorption and color strength at the optimum dyeing time (64min), temperature (78 o C), and dye concentration (61.62mg/ml) were 63.194% and 7.391 respectively. The washing fastness (color durability) of FeSO4 mordanted cotton fabric dyed by Agave sisalana natural dye was good up to 10 washing cycle and within acceptability tolerance limit.