This preprint shows that Enterocin C (EntC), a two-peptide bacteriocin from Enterococcus faecalis, kills target bacteria by binding the membrane-embedded lipid-carrier recycling protein BacA with high affinity, forming a cooperative EntC1–EntC2–BacA complex that permeabilizes the membrane. Flow Induced Dispersion Analysis (FIDA), on a Fida 1 instrument, was used to measure the binding affinity (Kd) of EntC1 and EntC2 to BacA reconstituted in detergent micelles, revealing nanomolar-range, cooperative binding and confirming selectivity for BacA over related membrane proteins. It is an example of using FIDA to quantify peptide binding affinity to membrane protein targets in a detergent-solubilized, near-native environment.