The clinically important vancomycin antibiotic inhibits the growth of pathogens such as Staphylococcus aureus by blocking cell wall synthesis through specific recognition of nascent peptidoglycan terminating in D-Ala-D-Ala. Here, we demonstrate the ability of single-molecule atomic force microscopy with antibiotic-modified tips to measure the specific binding forces of vancomycin and to map individual ligands on living bacteria. The single-molecule approach presented here provides new opportunities for understanding the binding mechanisms of antibiotics and for exploring the architecture of bacterial cell walls.