<p>Biofilms, complex microbial communities embedded in extracellular polymeric substances (EPS), pose significant challenges across medical, industrial and environmental sectors due to their enhanced resistance to antibiotics and chemical agents. Traditional treatments often fail to combat biofilm-associated infections, leading to increased healthcare costs and mortality rates. This comprehensive review explores the potential of natural anti-biofilm agents derived from plants, microorganisms, marine organisms and other natural sources as alternative strategies. Plant-derived compounds, microbial products and marine bioactive substances exhibit promising anti-biofilm properties through various mechanisms, including inhibition of microbial adhesion, quorum sensing (QS) interference and EPS disruption. The review highlights the efficacy of these agents in both in vitro and in vivo studies and discusses their potential applications in industries such as medicine, food processing and water treatment. Despite the promising results, challenges such as standardization, biocompatibility and environmental impact remain. Future research should focus on optimizing these natural agents and exploring synergistic combinations to enhance their efficacy. This review underscores the importance of sustainable and interdisciplinary approaches in developing effective, eco-friendly anti-biofilm strategies for diverse applications.</p>