Many Gram-negative bacteria employ N-acyl homoserine lactones (AHLs) as signal molecules for quorum sensing. The binding of AHLs to their target LuxR-type receptor proteins can effect changes in growth, virulence, and other phenotypes. LuxR-type receptors therefore present attractive pharmaceutical targets for control of bacterial pathogenesis. Here, we present X-ray crystallographic and computational evidence that the conformation of free AHLs is biased away from the conformation observed when bound to their cognate receptor due to the influence of an n→π* interaction. In this n→π* interaction, the p-type lone pair (n) of the N-acyl oxygen overlaps with the π* orbital of the lactone carbonyl group. This overlap results in the release of approximately 0.64 kcal/mol of energy. We also show that this interaction can be attenuated by installing electron-withdrawing groups on the N-acyl chain. Modulating this previously unappreciated interaction could present a new avenue toward effective inhibitors of bacterial quorum sensing.