Considering the importance of developing environmentally friendly dyeing processes and utilizing renewable natural resources, this study investigated the dyeing of wool fabric using red onion peel extract as a bio-based and readily available source derived from agricultural waste. The aim of this study was to develop a sustainable approach for wool dyeing by evaluating the effects of mordant type and mordanting method on the colorimetric and fastness properties of dyed samples, as well as assessing the potential of plant-based mordants as bio-based alternatives in the dyeing process. For this purpose, the dyeing conditions were first optimized stepwise based on color strength. The results showed that a dye concentration of 80% owf, pH 3, a dyeing temperature of 80 °C, and a dyeing time of 60 min were suitable conditions for wool dyeing. Under these conditions, the color strength of the un-mordanted sample reached 11.54. The effects of metallic and plant-based mordants were then evaluated using three mordanting methods, namely pre-mordanting, meta-mordanting, and post-mordanting, in terms of the colorimetric and fastness properties of the dyed samples. In the pre-mordanting method, Sn produced the highest color strength at the wavelength of maximum absorption, with a value of 21.95. In the post-mordanting method, Cr and Fe produced the highest average color strength values, 11.58 and 11.32, respectively, and generated the darkest shades. Plant-based mordants generally had a more limited effect on increasing color depth; however, some of them, including date kernel, gall oak, walnut peel, and nettle, were effective in selectively improving washing, light, or sublimation fastness. Overall, the results indicate that the selection of mordant and mordanting method should be made according to the final dyeing objective, including color strength, desired shade, and the required type of fastness.