The therapeutic efficiency and topical performance of drug-containing microcapsules varied when the drugs existed in an internal oil phase or an internal aqueous phase within the wall shell or wall matrix of microcapsules. In this study, chitosan-based (oil-in-water) and agar-gelatin-based (water-in-oil) microencapsulation systems containing berberine were applied to cotton fabrics to provide an anti-Staphylococcus aureus activity for textile materials. The berberine microcapsule-treated cotton samples were subjected to various washing cycles and their surface morphology, chemical compositions and antibacterial property were investigated after washing. The SEM images and Fourier transform infrared analysis showed that the amount of microcapsules on cotton samples decreased gradually with an increase in washing cycles. After 20 washing cycles, the cotton fabrics with agar-gelatin (water-in-oil) microcapsules containing berberine still exhibited the anti-S. aureus activity. However, the chitosan-based (oil-in-water) system did not show any growth inhibition towards S. aureus but only in the contact areas.