We previously reported a series of bis-sulfonamido-2-phenylbenzoxazole (PBZ) inhibitors of GroEL/ES and HSP60/10 chaperonin systems that exhibited potent antibiotic effects against bacteria and Trypanosoma brucei parasites, and chemotherapeutic effects against colorectal cancer cells. However, the mechanisms of action of these inhibitors at the protein level and their relative impact on folding in bacterial versus human cells remained unknown. In this study, cryoEM analyses identified a set of 7-fold symmetrical inhibitor binding sites at the GroEL ring-ring interface (RRI). In addition, LC-MS/MS analyses identified two sets of binding sites at the apical domains and the ATP pockets. These findings, coupled with results from biochemical assays evaluating a small panel of PBZ-based inhibitors, allowed the development of a unique mechanistic model that explains how PBZ binding at the three sites modulates the ATPase and client protein folding functions of E. coli GroEL/ES. Screening of our diversity set of structurally distinct GroEL/ES inhibitors highlighted the unique mechanism of PBZ compounds binding to the RRI and disrupting the intra- and inter-ring allostery governing chaperonin function. We further developed complementary intravital reporter assays to measure the targeting of GroEL/ES in S. aureus bacteria and HSP60/10 in the mitochondria of human embryonic kidney (HEK) 293 cells. Compound screening using these assays support the differential engagement of PBZ analogs with chaperonins in bacteria and/or human cells. Together, these results provide a more detailed molecular basis for the findings from our companion study by Godek et al. that elucidated on-target functions of our lead analog, PBZ1038 (compound 1), against GroEL in E. coli. The inhibition model developed here will be invaluable for interpreting the mechanisms of new inhibitors and generating more potent and selective analogs as antibacterial candidates.