We report an approach to attach first-generation donor-acceptor Stenhouse adducts (DASAs) onto cotton fabric. First-generation DASAs were immobilized onto cotton fabric samples using the following two-step approach: (1) Secondary amines were created via the functionalization of N, N-methyl(2-bromo-ethyl) ammonium bromide on the cotton substrates. (2) DASA compounds 5-(furan-2-ylmethylene)-2,2-dimethyl-1,3-dioxane-4,6-dione were immobilized on the functionalized cotton substrates from the secondary amines inducing ring-opening of Meldrum’s activated furans. Four types of cotton were used in this study: bleached desized cotton (BDC), combed cotton batiste (CCB), interlock knit tubular (IKT), and cotton twill (CT). Four environmental exposure conditions of visible light irradiation and relative humidity (RH) were evaluated to study the color difference (∆E) as the DASA compounds isomerized from a colored, linear form to a colorless cyclic form and vice versa. These conditions included light irradiation and 18% RH (L18), light irradiation and 65% RH (L65), no light irradiation or dark and 18% RH (D18), dark and 65% RH (D65). The color changes in the cotton samples containing DASA were also induced with water and tested for thermal reversion. FTIR confirmed the attachment of DASAs on the cotton substrates which supports a visual assessment as all cotton samples shifted to a pink color after DASA immobilization. The color differences were small and hence difficult to be perceived by a human eye, so we used colorimetry measurements to quantitatively determine color differences in the L, ab color space. Colorimetric linear mixed models were constructed to analyze the color space data across fixed effects such as RH, exposure to visible light irradiation, and cotton type. Amongst those fixed effects, light irradiation and humidity were found to elicit the greatest color changes.