In order to provide a sustainable substitute for synthetic dyes, this study investigates the environmentally friendly coloration of cotton fabric utilizing lemon juice as a biomordant and holy basil leaves extracts as a natural dye. Twelve samples of cotton single jersey fabric were mordanted with lemon juice and then dyed with holy basil leaves extracts under acidic, neutral, and alkaline conditions at 40, 60, 80, and 100 °C. The results showed that the fabric dyed in an alkaline medium at 100 °C had the best color uptake and the highest color strength (K/S), as well as the best fastness to rubbing, washing, perspiration, and light fastness properties. The scanning electron microscopy (SEM) test results empirically validate the dyeing process, as the highest surface scratches were observed on colored cotton fabric dyed in the alkaline medium at 100 °C. Thermal analysis (Tg, DTg, DTA) reveals that the dyed fabric has improved thermal stability, with delayed decomposition and reduced weight loss compared to the undyed fabric, indicating a protective interaction between the dye and cotton fibers. The Fourier-transform infrared (FTIR) spectroscopy analysis also confirmed the interaction of cotton fabric with lemon juice mordant and holy basil leaf extracted natural dye particles. X-ray diffraction (XRD) analysis demonstrates that the dyeing process preserves the crystalline cellulose structure while introducing less crystalline or amorphous components from the basil extract. This research promotes the potential of holy basil leaf extract and lemon juice as a natural dyeing system, particularly effective in an alkaline medium at 100 °C, promoting sustainable practices in textile coloration.