This research focuses on flavonoid-based natural dyes for the coloration and antibacterial finishing of wool fabrics. In the present study, we accessed the suitability of biocolorants from Gingko biloba L. waste/fallen leaves extract to develop colored antibacterial wool fabrics to reduce the environmental pollution and dependency of the textile industry on the synthetic antibacterial agents. Specifically, we present a novel method to counteract the growing antibiotic resistance using G. biloba L. leaves extract as a potent antibacterial agent in conjunction with ampicillin. Natural colorants were extracted in 50% aqueous ethanol at 70 °C for 60 min under acid conditions of pH 3. Before dyeing procedures, the extracted dye powder was assessed for thermal stability by TGA analysis. Structural characterization of the extracted dye was done by UV–visible and FT-IR spectral analysis. Optimal dyeing results were achieved at 90 °C for 70 min with a dye concentration of 50% (o.w.f.). Additionally, dye adsorption (Isotherm constants) and dye performances were effectively reported during this study. Pseudo-second order and sips isotherm model provided the best fitting of experimental data with high regression coefficient (R2 = 0.99). Valuated indicators (color strength (K/S), color characteristics, and fastness properties) of dyed wool fabric were effectively reported in response to washing pretreated with different metallic and natural mordants. The antibacterial potential of extracted natural dye was enhanced in combination with ampicillin (synergism) which can be correlated with the significant reduction in the corresponding MIC values. Fe2+ and Sn2+ salts demonstrated negative effects on the antibacterial performance of the dyed wool fabric, whereas most of the biomordants showed enhanced antibacterial performances. The effective use of the extracted yellow dye from G. biloba L. fallen/waste leaves in textile dyeing and finishing will enhance the industrial applications of the green waste over its normal applications. Display omitted •Waste leaves of G. biloba L. have been used for natural dye production.•Natural dye was extracted in 50% aqueous ethanol solution under acidic conditions.•Adsorption kinetic showed pseudo-second-order fitting.•MIC of dye extract was enhanced in conjunction with ampicillin.•Biomordants were more effective in enhancing antibacterial properties.