Bacterial biofilms form a thick, highly structured protective layer that shields bacterial cells from environmental stress, including antibiotic exposure. Several key factors contribute to biofilm persistence, with persister cells and the extracellular polymeric matrix playing crucial roles. Persister cells are metabolically dormant, non-dividing subpopulations that exhibit exceptional tolerance to antimicrobial agents, allowing biofilms to survive even in harsh conditions. The stringent response system, regulated by RelA and SpoT genes, is a critical mechanism in persister cell formation, primarily through the production of the signaling molecule guanosine tetraphosphate/pentaphosphate. This molecule acts as a global regulator, controlling bacterial metabolism and stress responses, thereby enhancing biofilm resilience. The presence of persister cells in biofilms presents significant challenges in both clinical and industrial settings, contributing to persistent infections and antimicrobial resistance. This chapter explores the formation, mechanisms, and molecular perspectives of persister cells within biofilms, providing insights into their role in biofilm-associated infections and potential therapeutic interventions.