Novel fluoroquinolone derivatives substituted with a 2-thioalkyl moiety, with and without a concomitant 3-carboxylate group, were synthesized to evaluate the effect of C-2 thioalkyl substituents on gyrase binding and inhibition. The presence of a 2-thioalkyl group universally decreased activity as compared to parent fluoroquinolones. However, with derivatives of moxifloxacin the presence of either a 2-thioalkyl group or a 3-carboxylate moiety increased activity over the 2,3-unsubstituted derivative. Energy minimization of structures provides an explanation for relative activities of fluoroquinolones having a C-2 thio moiety.