Antibiotic resistance of pathogens is among the major concerns in various medical applications. Therefore, the search for the novel antimicrobial agents that could prevent pathogen’s resistance, while maintaining efficient treatment, is one of the most important issues for biomedicine nowadays. One of the relevant methods for the development of functional non-woven materials possessing antimicrobial properties is the use of ozone and ozonolysis products for the modification of fibrous materials. This approach has recently attracted both academic and industrial interest and has found various biomedical applications. Several methods providing antimicrobial properties to textiles using ozone or ozonolysis products were proposed, including encapsulation and/or direct introduction of ozone-generated antimicrobial agents into the fibrous polymer matrix and ozone treatment of non-woven fiber materials. For the latter, the ozonolysis products are uniformly distributed predominantly on the polymer surface but could be also formed inside the polymer bulk due to ozone diffusion through the amorphous areas or defects. It was found that ozone modification of fibrous materials could lead to increase in hydrophilicity and improvement in their functional properties (smoothness, elasticity, strength, antimicrobial activity). In this chapter, various aspects of ozone modification of non-woven fiber materials for biomedical applications are reported and discussed.