Functionalized textiles can be used in wound management to reduce the microbial burden in the wound area, to prevent wound infections, and to avoid cross-contamination between patients. In the present study, a comprehensive in vitro approach to enable the assessment of antibacterial activity of functionalized textiles and cytotoxicity of cyclodextrin (CD)-complexes with chlorhexidine diacetate (CHX), iodine (IOD), and polihexanide (PHMB) is suggested to evaluate their properties for supporting optimal conditions for wound healing. For all β-CD-antiseptic functionalized cotton samples a strong antibacterial effect on the Gram-positive bacteria Staphylococcus aureus and Staphylococcus epidermidis as well as on the Gram-negative bacteria Klebsiella pneumoniae and Escherichia coli was proven. In addition, β-CD-CHX and β-CD-PHMB were effective against the yeast Candida albicans. The growth of Pseudomonas aeruginosa could be reduced significantly by β-CD-IOD and β-CD-PHMB. The established comprehensive testing system for determination of biocompatibility on human HaCaT keratinocytes is suitable for obtaining robust data on cell viability, cytotoxicity and mode of cell death of the β-CD-antiseptic-complexes. The promising results of the high antimicrobial activity of these functionalized textiles show the high potential of such materials in medical applications.