The development of new antibacterial therapeutic agents capable of halting microbial resistance is a chief pursuit in clinical medicine. Classes of antibiotics that target and destroy bacterial membranes are attractive due to the decreased likelihood that bacteria will be able to generate resistance to this mechanism. The amphipathic cyclic decapeptide, Tyrocidine A, is a model for this class of antibiotics. Tyrocidine A is composed of a hydrophobic and a hydrophilic face, allowing for insertion into bacterial membranes, creating porous channels and destroying membrane integrity. We have used a combination of molecular modeling and solid phase synthesis to prepare Tyrocidine A and analogues 1-8. The minimum inhibitory concentrations (MICs) of these compounds were determined for a host of gram positive species and E. coli as a representative gram negative bacterium. Analogues 2 and 5 demonstrated moderate 2- to 8-fold increases in antibacterial activity over the parent Tyrocidine A for a variety of pathogenic microbes (best MICs for E. coli 32 microg/mL and 2 microg/mL for most gram positives). Examination of the structure- activity relationship between the analogues demonstrated a preference for increased amphipathicity but did not show a clear preference for increasing hydrophilicity versus hydrophobicity in improving antibacterial activity. Of note, movement of positively charged lysine residues or neutral pentafluorophenyl residues to different positions within the cyclopeptide ring system demonstrated improvements in antibacterial activity.