Chitosan, a cationic polysaccharide derived from the deacetylation of chitin, is widely distributed in nature. Its antibacterial activity, biocompatibility, biodegradability, and non-toxicity have given it extensive uses in medicine, food, and cosmetics. However, the significant impact of variations in the physicochemical properties of chitosan extracted from different sources on its application efficacy, as well as the considerable differences in its antimicrobial mechanisms under varying conditions, limit the full realization of its biological functions. Therefore, this paper provides a comprehensive review of the structural characteristics of chitosan, its preparation methods from different sources, its antimicrobial mechanisms, and the factors influencing its antimicrobial efficacy. Furthermore, we highlight the latest applications of chitosan and its derivatives across various fields. We found that the use of microbial extraction shows promise as a new method for producing high-quality chitosan. By analyzing the different physicochemical properties of chitosan from various sources and the application of chitosan-based materials (such as nanoparticles, films, sponges, and hydrogels) prepared using different methods in biomedicine, food, agriculture, and cosmetics, we expect these findings to provide theoretical support for the broader utilization of chitosan.