The opportunistic pathogen Pseudomonas aeruginosa uses quorum sensing (QS) to regulate many of its virulence traits. It has a QS system based on N-acyl L-homoserine lactone (AHL) signal molecules that are produced by LuxI-type synthase enzymes and sensed by intracellular LuxR-type receptor proteins. We have developed several QS inhibitors (QSIs) that strongly inhibit LuxR-type proteins and can be used as mechanistic probes and potential antivirulence treatments. The research described in this thesis explores the mechanisms by which these QSIs function and the potential for resistance to develop to such antivirulence agents. Using competitive growth studies of P. aeruginosa QS mutants under infection-relevant conditions, we first demonstrated that two discrete obstacles impede the spread of QSI resistance: (1) a small number of QSI-resistant mutants cannot produce enough signal to induce QS, and (2) group-beneficial QS-regulated traits render QSI-resistant bacteria susceptible to cheating by QSI-sensitive neighbors. Having shown that resistance will likely not spread quickly to QSIs, we aimed to better understand how QSIs function in order to improve their potency. We tested the activity of several non-native QS modulators on site-directed mutants of the LasR QS receptor. Our data strongly suggested that the synthetic ligands bind LasR in an orientation analogous to its native signal molecule, N-(3-oxo)-dodecanoyl L-homoserine lactone (OdDHL), and that the interactions of their lactone head groups with Trp60 are important in governing whether they activate or inhibit LasR. This information can be leveraged to design more potent QSIs. Also, we hypothesized that active efflux decreases the potency of LasR inhibitors. Indeed, we found that the MexAB-OprM multidrug efflux pump was responsible for substantial losses in potency for every QSI tested except 5,6-dimethyl-2-aminobenzimidazole (DMABI), which may serve as a scaffold for the design of efflux-resistant QSIs. Lastly, since P. aeruginosa and many other microbes frequently live in polymicrobial communities, their QS circuits may be influenced by signals produced by neighboring bacteria. To that end, we characterized the ligand-activation specificity of LasR and a related protein, AbaR from Acinetobacter baumannii, and developed a new model to predict the promiscuity with which bacteria in polymicrobial environments respond to other QS signals.