Microorganisms can aggregate and organise into structured communities embedded within an exopolysaccharide-based matrix, which serves as a protective barrier and an functional environment around microbial cells. The formation of biofilms is widely recognised as a pivotal factor in bacterial virulence, impeding the efficacy of antimicrobial agents and hindering immune responses, whilst concomitantly contributing to the development of antimicrobial resistance and the onset of persistent infections. Biofilm formation is a tightly regulated and dynamic process, controlled by quorum sensing mechanisms and profoundly influenced by enviromental factors and nutrient availability. The objective of this review is to elucidate the significance of biofilms in clinical settings, with a particular focus on their role in the pathogenesis of infectious diseases. Particular attention is devoted to biofilm-associated infections related to invasive medical devices as well as to chronic conditions occurring in predisposed patients, including individuals with cystic fibrosis. This review also examines the structure of biofilms and the bacteria responsible for their production, as well as the latest strategies for combating antibiotic resistance in bacterial biofilms. Furthermore, the role of biofilm-associated infections is analysed, with a focus on recent advancements in biofilm pathogenesis, diagnostic challenges and the necessity for innovative therapeutic strategies to disrupt biofilm integrity.