Natural biopolymers as lignin, a common plant biopolymer, look interesting as antimicrobial agents against the pathogenic microorganisms. This study aims to produce eco-friendly products by extracting lignin from two Egyptian cotton cultivars, Giza 86 and Giza 90, using the organosolv technique and transferring them into lignin nanoparticles then to determine lignin and lignin nanoparticles bioactivities and to treat textiles for medical applications against seven harmful bacteria and five mycotoxigenic fungi. OLNPs were studied using Zetasizer, Fourier Transformer Infrared Spectroscopy (FTIR), and Transmission Electron Microscope (TEM). The results revealed that lignin and lignin nanoparticles had a greater effect on bacteria than fungi. Penicillium verrucosum exhibited the greatest inhibition zone (15.5±1.32) for OLNP86, while Salmonella typhi exhibited the greatest inhibition zone (14.7±1.28) for OLNP86. Similarly, textiles treated with lignin and lignin nanoparticles showed a clear effect on bacteria and fungi under study, where Penicillium verrucosum exhibited the greatest inhibition zone (30.7±1.76), and Pseudomonas aeruginosa exhibited the greatest inhibition zone (36.3 ±0.58) for OLNP86. The current study shows that OLNP's antioxidant and antimicrobial characteristics can be further utilized in medical textiles, which can be used for patients with conditions such as diabetes, high blood pressure, and kidney failure who cannot tolerate antibiotics.