One interference mechanism of bacterial competition is the production of antibiotics. Bacteria exposed to antibiotics can resist antibiotic inhibition through intrinsic or acquired mechanisms. Here, we performed a coevolution experiment to understand the long-term consequences of antibiotic production and antibiotic susceptibility for two environmental bacterial strains. We grew five independent lines of the antibiotic-producing environmental strain,Burkholderia thailandensisE264, and the antibiotic-inhibited environmental strain,Flavobacterium johnsoniaeUW101, together and separately on agar plates for 7.5 months (1.5 month incubations), transferring each line five times to new agar plates. We observed that theF. johnsoniaeancestor could tolerate theB. thailandensis-produced antibiotic through efflux mechanisms, but that the coevolved lines had reduced susceptibility. We then sequenced genomes from the coevolved and monocultureF. johnsoniaelines, and uncovered mutational ramifications for the long-term antibiotic exposure. The coevolved genomes fromF. johnsoniaerevealed four potential mutational signatures of reduced antibiotic susceptibility that were not observed in the evolved monoculture lines. Two mutations were found intolC: one corresponding to a 33 bp deletion and the other corresponding to a nonsynonymous mutation. A third mutation was observed as a 1 bp insertion coding for a RagB/SusD nutrient uptake protein. The last mutation was a G83R nonsynonymous mutation in acetyl-coA carboxylayse carboxyltransferase subunit alpha (AccA). Deleting the 33 bp fromtolCin theF. johnsoniaeancestor reduced antibiotic susceptibility, but not to the degree observed in coevolved lines. Furthermore, theaccAmutation matched a previously described mutation conferring resistance toB. thailandensis-produced thailandamide. Analysis ofB. thailandensistransposon mutants for thailandamide production revealed that thailandamide was bioactive againstF. johnsoniae,but also suggested that additionalB. thailandensis-produced antibiotics were involved in the inhibition ofF. johnsoniae. This study reveals how multi-generational interspecies interactions, mediated through chemical exchange, can result in novel interaction-specific mutations, some of which may contribute to reductions in antibiotic susceptibility.