In this work, linoleic acid (LA), a long chain fatty acid bearing 18 carbons and two double bonds, inhibited hydrogen consumption in a mixed anaerobic culture acclimated to glucose. At pH=7.6 , a metabolic shift from methane to hydrogen formation was observed in cultures maintained at 37°C and fed 5,000 mg L−1 glucose in the presence of 500–2,000 mg L−1 LA. The hydrogen yield increased with increasing LA levels while the quantity of methane decreased. The major volatile fatty acids produced were acetate and butyrate with greater levels observed in cultures fed with LA. Acetate, butyrate, and hydrogen accumulation suggests inhibition of aceticlastic methanogens, hydrogenotrophic methanogens, and butyrate degrading microorganisms, respectively, in the presence of LA. A maximum hydrogen yield of 1.71±0.22 mole mole−1 glucose was observed only when glucose was reinjected into cultures receiving 2,000 mg L−1 LA plus 5,000 mg L−1 glucose.