The objective of this study was to evaluate the ability of spent coffee extract to enhance the antioxidant and antimicrobial properties of cotton fabrics. Spent coffee collected after brewing using an espresso machine was extracted with 60% methanol solution, and the extract was applied to cotton fabrics through a pad-dry-cure process. Of the compounds identified in the spent coffee extract, caffeine (381.09 μg/mL) was the most abundant, followed by chlorogenic acid (213.93 μg/mL) and trigonelline (155.20 μg/mL). The cotton fabrics finished with the spent coffee extract showed a slight increase in yellowness. In addition, the antioxidant activity increased proportional to the concentration of the spent coffee extract. The cotton fabrics finished with the spent coffee extract inhibited the growth of Gram-positive Staphylococcus aureus but did not significantly affect the growth of Gram-negative Klebsiella pneumoniae. These results show that spent coffee can be used as a natural finishing agent for cotton to improve the antioxidant and antimicrobial properties of the fabric.