Recently we described a novel di-benzene-pyrylium-indolene (BAS00127538) inhibitor of Lipid II. BAS00127538 (1-Methyl-2,4-diphenyl-6-((1E,3E)-3-(1,3,3-trimethylindolin-2-ylidene)prop-1-en-1-yl)pyryl-1-ium) tetrafluoroborate is the first small molecule Lipid II inhibitor and is structurally distinct from natural agents that bind Lipid II, such as vancomycin. Here, we describe the synthesis and biological evaluation of 50 new analogs of BAS00127538 designed to explore the structure-activity relationships of the scaffold. The results of this study indicate an activity map of the scaffold, identifying regions that are critical to cytotoxicity, Lipid II binding and range of anti-bacterial action. One compound, 6jc48-1, showed significantly enhanced drug-like properties compared to BAS00127538. 6jc48-1 has reduced cytotoxicity, while retaining specific Lipid II binding and activity against Enterococcus spp. in vitro and in vivo. Further, this compound showed a markedly improved pharmacokinetic profile with a half-life of over 13 hours upon intravenous and oral administration and was stable in plasma. These results suggest that scaffolds like that of 6jc48-1 can be developed into small molecule antibiotic drugs that target Lipid II.