In this study, a novel green approach was adopted to develop antimicrobial polyester fabric using sustainable biopolymers (chitosan/derivative) as eco-friendly antimicrobial agents via the resource efficient supercritical CO2 (scCO2) dyeing route in a single step. Polyester fabric was dyed with a small amount of dye (0.4% owf) in the presence of chitosan/derivative (3% owf) in scCO2 at 120 °C, 25 MPa for 1 h. The success of chitosan/derivative impregnation was confirmed by Fourier Transform Infrared (FTIR), Zeta Potential (ζ), Scanning Electron Microscopy (SEM), and Water Contact Angle (WCA) measurements. According to the result, excellent color strength and fastness properties were obtained and the treated samples also reduced 75 − 93% of Escherichia coli (ATCC 25922) bacteria within one hour. This suggests that the dye and chitosan/derivative had no adverse effect on each other, proving compatibility. This new approach would help to reduce the cost of production and environmental pollution associated with the conventional textile finishing processes.