Vancomycin (Van) is a glycopeptide antibiotic commonly used as a last resort for treating life-threatening infections caused by multidrug-resistant bacterial strains, such as and spp. However, its effectiveness is currently limited due to the rapidly increasing number of drug-resistant clinical strains and its inherent cytotoxicity and poor penetration into cells and specific regions of the body, such as the brain. One of the most promising strategies to enhance its efficacy appears to be the covalent attachment of cell-penetrating peptides (CPPs) to the Van structure. In this study, a series of vancomycin conjugates with CPPs-such as TP10, Tat (47-57), PTD4, and Arg-were designed and synthesized. These conjugates were tested for antimicrobial activity against four reference strains (, , , and ) and two clinical drug-resistant strains: methicillin-resistant and vancomycin-resistant . In addition, cytotoxicity tests (using a human fibroblast cell line) and blood-brain barrier (BBB) permeability tests (using a parallel artificial membrane permeability assay-PAMPA-BBB assay) were conducted for selected compounds. Our research demonstrated that conjugation of Van with CPPs, particularly with Tat (47-57), Arg, or TP10, significantly enhances its antimicrobial activity against Gram-positive bacteria such as and spp., reduces its cytotoxicity, and improves its access to brain tissues. We conclude that these findings provide a strong foundation for the design of novel antimicrobial agents effective in treating infections caused by drug-resistant staphylococcal and enterococcal strains, while also being capable of crossing the BBB.