Fatty acids (FAs) can be used as carbon and energy source by most bacteria. FAs are very diverse and show variations in aliphatic chain length, degree and kind of branching, and number of double bonds. After their import in the cell and their activation by a thioester link to Coenzyme A, fatty acids are degraded by the β-oxidation machinery constituted of the Fad proteins. The core enzymes of the β-oxidation machinery can degrade most FAs, except for those FAs that bear an unsaturation at an even-numbered carbon. Such FAs include arachidonic acid or linoleic acid, which are essential FAs in the mammalian diet. We studied the role of the 2,4-dienoyl-CoA reductase FadH in E. coli metabolism of FAs. We showed that fadH is essential for growth on linoleic acid and that Cys residues acting as ligands for FadH-bound [Fe-S] cluster are essential for activity in vivo. Moreover, we showed that when it is mixed with other FAs, linoleic acid prevents growth of the fadH mutant. These results suggest a specific metabolic burden imposed by linoleic acid degradation, which might prove important in the context of the natural gut environment with mixed FA composition. Eukaryotes also use 2,4-dienoyl-CoA reductases for β-oxidation of FAs in mitochondria but these eukaryotic enzymes belong to a different family than FadH, with different co-factors equipment and mechanism. Yet, we showed that eukaryotic 2,4-dienoyl-CoA reductases can complement the E. coli fadH mutant for growth on linoleic acid and for relief of linoleate mediated jamming of the β-oxidation. Altogether these studies demonstrate the key role of prokaryotic and eukaryotic 2,4-dienoyl-CoA reductases in a complex environment containing mixtures of saturated and unsaturated FAs, as found in the gut or in the surrounding of cancer cells.