bEscherichia coli has been used as a platform host for studying the production of free fatty acids (FFA) and other energy-dense compounds useful in biofuel applications. Most of the FFA produced by E. coli are found extracellularly. This finding suggests that a mechanism for transport across the cell envelope exists, yet knowledge of proteins that may be responsible for export remains incomplete. Production of FFA has been shown to cause cell lysis, induce stress responses, and impair basic physiological processes. These phenotypes could potentially be diminished if efflux rates were increased. Here, a total of 15 genes and operons were deleted and screened for their impact on cell viability and titer in FFA-producing E. coli. Deletions of acrAB and rob and, to a lower degree of statistical confidence, emrAB, mdtEF, and mdtABCD reduced multiple measures of viability, while deletion of tolC nearly abolished FFA production. An acrAB emrAB deletion strain exhibited greatly reduced FFA titers approaching the tolC deletion phenotype. Expression of efflux pumps on multicopy plasmids did not improve endogenous FFA production in an acrAB ؉ strain, but plasmid-based expression of acrAB, mdtEF, and an mdtEF-tolC artificial operon improved the MIC of exogenously added decanoate for an acrAB mutant strain. The findings suggest that AcrAB-TolC is responsible for most of the FFA efflux in E. coli, with residual activity provided by other resistance-nodulation-cell division superfamily-type efflux pumps, including EmrAB-TolC and MdtEF-TolC. While the expression of these proteins on multicopy plasmids did not improve production over the basal level, their identification enables future engineering efforts.