In contemporary times, a notable surge of attention has been directed toward the incorporation of pharmaceutical-derived antibiotic agents in response to the textile industry's demands. This paradigm shift arises from the environmental consequences associated with the application of synthetic counterparts. Within the scope of this inquiry, an in-depth exploration was undertaken to scrutinize the intrinsic antibacterial attributes inherent to organic cotton fabric (OCF) following treatment involving the integration of antibiotics, particularly Nystatin. The core objective underpinning this endeavour was the comprehensive evaluation of Nystatin's potential as an antibacterial agent tailored for textile applications. To assess the ramifications of this treatment, the treated OCF underwent rigorous testing against two distinct bacterial strains, specifically Escherichia coli and Staphylococcus aureus. The well-established agar disc diffusion method was employed for this purpose. The outcomes gleaned from these meticulous experiments unequivocally underscored the remarkable antibacterial efficacy showcased by the treated fabric. This was manifested through the emergence of discernible zones of inhibition (ZOI), with measurements averaging 12.23 ± 0.97 mm against E. coli and 14.43 ± 0.78 mm against S. aureus before any washing intervention was conducted.