An efficient synthetic route was developed for the large-scale preparation of (±)-Yanglingmycin and its analogues. Three series of derivatives of (±)-Yanglingmycin were synthesized and the structures of all compounds were elucidated by analyses of NMR and ESI-MS spectra data. Moreover, their antibacterial activities against seven species of bacteria were systematically evaluated by the micro-broth dilution method, most of which displayed considerable activity. It was worth noting that compounds 5b, 5c, 5d, 6g, and 7 were found to be the most promising leading candidates, with peak MIC values of 0.98 μg·mL(-1) for Bacillus subtilis, which is superior to positive controls (MIC = 3.91 μg·mL(-1)). The above results might lay the firm foundation for the design and synthesis of novel antibacterial drugs based on (±)-Yanglingmycin.