The Resistance-Nodulation-Division (RND) efflux pump superfamily is pervasive among Gram-negative pathogens and contributes extensively to clinical antibiotic resistance. The opportunistic pathogenPseudomonas aeruginosacontains 12 RND-type efflux systems, with four contributing to resistance including MexXY-OprM which is uniquely able to export aminoglycosides. At the site of initial substrate recognition, small molecule probes of the inner membrane transporter (e.g., MexY) have potential as important functional tools to understand substrate selectivity and a foundation for developing adjuvant efflux pump inhibitors (EPIs). Here, we optimized the scaffold of berberine, a known but weak MexY EPI, using anin-silicohigh-throughput screen to identify di-berberine conjugates with enhanced synergistic action with aminoglycosides. Further, docking and molecular dynamics simulations of di-berberine conjugates reveal unique contact residues and thus sensitivities of MexY from distinctP. aeruginosastrains. This work thereby reveals di-berberine conjugates to be useful probes of MexY transporter function and potential leads for EPI development.