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Chitosan is a naturally originating product that can be applied in many areas due to its biocompatibility, biodegradability, and nontoxic properties. The broad-spectrum antimicrobial activity of chitosan offers great commercial potential for this product. Nevertheless, the antimicrobial activity of chitosan varies, because this activity is associated with its physicochemical characteristics and depends on the type of microorganism. In this review article, the fundamental properties, modes of antimicrobial action, and antimicrobial effects-related factors of chitosan are discussed. We further summarize how microorganisms genetically respond to chitosan. Finally, applications of chitosan-based biomaterials, such as nanoparticles and films, in combination with current clinical antibiotics or antifungal drugs, are also addressed.