Florfenicol (FF) is a widely used antimicrobial in veterinary medicine because of its broad antimicrobial activity, although it has certain limitations and raises concerns about the development of antimicrobial resistance genes. These limitations highlight the need to explore novel drug with controlled release systems to enhance the therapeutic efficacy of FF, while minimizing the potential for resistance development. This study introduces an innovative approach for the design, synthesis, and evaluation of lignin-poly(lactic-co-glycolic) acid (PLGA)-FF nanoparticles. By leveraging the properties of PLGA and lignin, this study aimed to augment the solubility, stability, and bioavailability of FF, thereby enabling dosage reduction and consequently diminishing the likelihood of resistance emergence and other limitations. Lignin-PLGA nanoparticles encapsulating FF were synthesized and characterized to assess their physicochemical properties, such as particle size, zeta potential, and drug loading efficiency. The release profile, antimicrobial efficacy, and cytotoxicity were evaluated. Comparative analyses with standard FF formulations were performed to ascertain the superior performance and potential benefits of the nanoparticle-based antimicrobials. Our findings indicate that the synthesized lignin-PLGA nanoparticles exhibited favorable drug delivery attributes, including a controlled and sustained release mechanism, significantly enhanced antimicrobial activity at reduced concentrations relative to free FF, with minimal cytotoxic effects. Importantly, the nanoparticle system inhibited bacterial biofilm formation, which is a key factor in the onset and spread of antimicrobial resistance. These findings underscore the potential of integrating biodegradable polymers with natural compounds to forge innovative pathways in drug delivery, addressing critical challenges in veterinary medicine.