Eucalyptus essential oils and extracts are widely recognized for their antimicrobial, antioxidant, insecticidal, anti-inflammatory, and aromatizing properties, making them highly valuable across pharmaceuticals, cosmetics, and textiles. For the design of biomedical textiles, cotton is favored for its mechanical strength, porosity, and biodegradability, but its vulnerability to microbial action limits its applications. To address this issue, natural compounds like eucalyptus essential oils and extracts have been prioritized over synthetic agents to enhance their antimicrobial properties. While bioactive textiles using essential oils have been well-documented, the incorporation of eucalyptus leaf extracts with different surface modifications of cotton remains largely unexplored. In this line, this study investigated pretreatment approaches to improve the uptake and uniformity of eucalyptus extract on cotton fibers. To achieve this, chitosan (CH), a cationic agent, and alum were applied to the cotton fabric to promote stronger electrostatic interactions and improve the binding of extract's bioactive components. The functionalized fabrics were tested for antimicrobial activity against , , and , antioxidant properties, and cytotoxicity using human keratinocytes. Eucalyptus-functionalized cotton, particularly with CH pretreatment, exhibited strong antibacterial activity against Gram-positive bacteria and enhanced antioxidant capacity in the DPPH assay. No cytotoxicity was detected with 8 h of exposure, but potential effects were observed after 24 h, indicating the need for further evaluation of long-term safety. These findings highlight the potential of eucalyptus-functionalized textiles for personalized clothing aimed at managing skin conditions linked to microbiota dysregulation, emphasizing the need for optimized functionalization and biocompatibility evaluation.