The main purpose of this work is to obtain a cotton-based textile material functionalized with L-cysteine (L-cys) to achieve an antimicrobial effect with potential application in biomedical, geriatric or pediatric textiles. The binding capacity of L-cys to cotton fibres was assessed through different functionalization strategies--surface activation and exhaustion processes. A subsequent analysis of the possible antibacterial action against Staphylococcus aureus and Klebsiella pneumoniae was performed according with the Japanese International standard (JISL, 2008). To determine the mechanism of action of L-cys on the selected strains, flow cytometry was used. The results revealed that the exhaustion process was performed with success to confer bioactivity to the treated fabric, as assessed by an effective antibacterial effect against both Gram-positive and Gram-negative bacteria, and successfully linkage of L-cys was observed via FTIR with a durable effect demonstrated after the washing tests (fastness to washing). It was also observed that L-cys exerts a bacteriostatic effect against both bacterial strains, since there were alterations in the metabolic activity of the microorganisms after the application of the bioactive textile which was shown by the CTC (cyanoditolyl tetrazolium chloride) staining used in flow cytometry. This study shows a new and successful biotechnological process to develop antibacterial textiles through the functionalization of cotton fibres with L-cys which presents a broad range of applications in healthcare, since L-cys is a natural antibacterial compound, non-toxic and affects pathogenic bacteria related to hospital infections.