It is an urgent need to tackle the global crisis of multidrug-resistant bacterial infections. We report here an innovative strategy for large-scale screening of new antibacterial agents using a whole bacteria-based DNA-encoded library (DEL) of vancomycin derivatives via peripheral modifications. A bacterial binding affinity assay was established to select the modification fragments in high-affinity compounds. The optimal resynthesized derivatives demonstrated excellently enhanced activity against various resistant bacterial strains and provided useful structures for vancomycin derivatization. This work presents the new concept in a natural product-templated DEL and in antibiotic discovery through bacterial affinity screening, which promotes the fight against drug-resistant bacteria.