Antibiotic-resistant and biofilm-associated infections constitute a rapidly growing issue. Use of the last resort antibiotic vancomycin is under threat, due to the increasing appearance of vancomycin resistant bacteria as well as the formation of biofilms. Herein, we report a series of novel vancomycin derivatives carrying thiol- and disulfide-containing moieties. The new compounds exhibited enhanced antibacterial activity against a broad range of bacterial strains, including vancomycin resistant microbes and Gram-negative bacteria. Moreover, all obtained derivatives demonstrated improved antibiofilm formation activity against VanB resistant Enterococcus compared to vancomycin. This work established a promising strategy for combating drug-resistant bacterial infections or disrupting biofilm formation and advances the knowledge on structural optimization of antibiotics with sulfur-containing modifications.