The formation of bacterial biofilms has increased the resistance of bacteria to various environmental factors and is tightly associated with many persistent and chronic bacterial infections. Herein we design a strategy conjugating florfenicol, an antibiotic commonly used in the treatment of streptococcus, with the antimicrobial biomaterial, chitosan oligosaccharides. The results demonstrated that the florfenicol-COS conjugate (F-COS) efficiently eradicated the mature Streptococcus hyovaginalis biofilm, apparently inhibiting drug resistance to florfenicol. A quantity of 250 μg/mL F-COS showed effective inhibitory activity against planktonic cells and biofilm of the bacteria, and a 4-fold improvement of the F-COS compared to unmodified florfenicol was observed. Furthermore, the conjugate showed a broad-spectrum activity against both Gram-positive and Gram-negative bacteria. It suggested that F-COS might have a potential for application in the treatment of biofilm-related infections.