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Healthcare-associated infections pose a significant global health challenge, negatively impacting patient outcomes and burdening healthcare systems. A major contributing factor to healthcare-associated infections is the formation of biofilms, structured microbial communities encased in a self-produced extracellular polymeric substance matrix. Biofilms are critical in disease etiology and antibiotic resistance, complicating treatment and infection control efforts. Their inherent resistance mechanisms enable them to withstand antibiotic therapies, leading to recurrent infections and increased morbidity. This review explores the development of biofilms and their dual roles in health and disease. It highlights the structural and protective functions of the EPS matrix, which shields microbial populations from immune responses and antimicrobial agents. Key molecular mechanisms of biofilm resistance, including restricted antibiotic penetration, persister cell dormancy, and genetic adaptations, are identified as significant barriers to effective management. Biofilms are implicated in various clinical contexts, including chronic wounds, medical device-associated infections, oral health complications, and surgical site infections. Their prevalence in hospital environments exacerbates infection control challenges and underscores the urgent need for innovative preventive and therapeutic strategies. This review evaluates cutting-edge approaches such as DNase-mediated biofilm disruption, RNAIII-inhibiting peptides, DNABII proteins, bacteriophage therapies, antimicrobial peptides, nanoparticle-based solutions, antimicrobial coatings, and antimicrobial lock therapies. It also examines critical challenges associated with biofilm-related healthcare-associated infections, including diagnostic difficulties, disinfectant resistance, and economic implications. This review emphasizes the need for a multidisciplinary approach and underscores the importance of understanding biofilm dynamics, their role in disease pathogenesis, and the advancements in therapeutic strategies to combat biofilm-associated infections effectively in clinical settings. These insights aim to enhance treatment outcomes and reduce the burden of biofilm-related diseases.