Daptomycin is effective in treating infections caused by antibiotic-resistant Gram-positive pathogens, including methicillin-resistant <i>Staphylococcus aureus</i> (MRSA), vancomycin-resistant <i>Enterococci</i> (VRE), and vancomycin-resistant <i>S. aureus</i> (VRSA). Due to its distinct mechanism of action toward multidrug-resistant bacteria, daptomycin provides an attractive structural motif to generate new daptomycin-based antibiotics to combat the problem of bacterial resistance. In this study, we used the total synthesis method to produce daptomycin analogues with a variety in terms of types and sites of modifications. Five classes of daptomycin analogues were synthesized, and the antimicrobial activities of the analogues were analyzed by several biological assays. From this study, we established a comprehensive structure-activity relationship of daptomycin which will lay the foundation for the further development of daptomycin-based antibiotics.