This study focused on effective natural colorants extraction from onion (Allium cepa L.) peel and applying the extracted dye on 100% organic cotton fabric assisted by ultrasonication (US) technique. Three techniques, pre-mordanting, simultaneous mordanting, and post-mordanting, were employed, using lemon juice as a mordant to assist the dye material for steady penetration and fixation into the pre-treated organic cotton fabric. The ISO standards were maintained in color fastness tests to compare the stability of dyed samples. Fastness properties showed moderate to good color change and staining for the post-mordanted sample. Fourier Transform Infrared Spectroscopy evidenced potential binding sites having stretched N–H and C–O vibration of alcohols, esters, ethers, carboxylic acids, anhydrides, and medium C–N stretching at 3330 cm−1 and 1028 cm−1, respectively. Within the visible range of 400–700 nm, the spectrophotometer test was carried out to obtain CIELAB and CIELCH color coordinates (CIE L*, a*, b*, C*, h*), color strength (K/S) values to ensure dye fixation together with after-dyeing behavior. The pre-mordanted sample showed comparatively better color strength (K/S) which was 2.6, and the post-mordanted sample showed better color fastness properties.