DNA origami nanostructures (DONs) have promising applications in biomedicine and biosensing, which often require their efficient binding to target cells. By immobilizing the glycopeptide antibiotic vancomycin on DONs, DON binding to Gram-positive and Gram-negative bacteria can be facilitated. Here, we investigate how this multivalent binding is affected by the number and arrangement of the vancomycin modifications on two-dimensional DONs. We find that for both Gram-positive Bacillus subtilis and Gram-negative Escherichia coli, binding increases with the number of vancomycin modifications per DON. In general, binding to E. coli is stronger than to B. subtilis, which may be attributed to differences in the architectures of the cell envelopes. Interestingly, for both bacteria, the total number of vancomycin modifications appears to be more important than their arrangement, as DONs with 18 vancomycin molecules on one side show similar binding as DONs with 18 vancomycin molecules distributed over both sides. This enables the attachment of multiple probe molecules to the vancomycin-free side of the DONs for enhancing detection efficiency without compromising binding affinity. These results may thus provide guidelines for the design and synthesis of vancomycin-modified DONs for antimicrobial drug delivery and pathogen detection.