A significant increase of microbial resistance to glycopeptides (especially vancomycin-resistant enterococci andStaphylococcus aureus) prompted researchers to design new semisynthetic glycopeptide derivatives, such as dual-action antibiotics that contain a glycopeptide molecule and an antibacterial agent of a different class. We synthesized novel dimeric conjugates of kanamycin A with glycopeptide antibiotics, vancomycin and eremomycin. Using tandem mass spectrometry fragmentation, UV, IR, and NMR spectral data, it was unequivocally proven that the glycopeptide is attached to the kanamycin A molecule at the position 1 of 2-deoxy-D-streptamine. New MS fragmentation patterns forN-Cbz-protected aminoglycosides were discovered. It was found that the resulting conjugates are active against Gram-positive bacteria, and some are active against vancomycin-resistant strains. Conjugates of two different classes can serve as dual-target antimicrobial candidates for further investigation and improvement.