Bacteria employ biofilm formation as a survival strategy, characterized by the self-assembly of cells into 3D architectures encapsulated in an extracellular polymeric substance (EPS) that results in reduced antibiotic efficacy, increased tolerance, and emergence of multidrug resistance phenotypes. To overcome this challenge, persistent efforts are directed toward developing cutting-edge approaches and agents that rejuvenate antibiotic efficacy, mitigate biofilm formation, and eradicate biofilm-associated bacterial infections. Within this framework, nanotechnology has emerged as a pivotal tool for developing innovative functional materials with tailored attributes, exhibiting substantial potential in addressing the global health challenge of antibiotic resistance and biofilm-associated infections. This updated review article provides a comprehensive overview, commencing with a thorough analysis of biofilm formation and its implications, followed by a critical evaluation of cutting-edge strategies derived from recent research advancements. Our discussion encompasses novel strategies, including traditional nanomaterials, micro-nanobubbles, multifunctional nanozyme-mimetic platforms, artificial phage-like structures, and sophisticated nano-microrobotic systems. Each strategy is assessed for its potential to effectively target biofilms, enhance antimicrobial penetration, and restore antibiotic susceptibility. We anticipate that this timely review will inform and inspire innovative research directions, focusing on the rational design and application of advanced nanomaterials for targeted biofilm modulation and efficacious treatment, thereby advancing healthcare solutions.