Ammonia and long chain fatty acids (LCFA) are two major inhibitors of the anaerobic digestion (AD) process. The individual inhibitory effect of each of these two inhibitors is well established; however, the combined co-inhibition effect has not been thoroughly assessed yet. In the current study, the ammonia-LCFA synergetic co-inhibition effect was investigated in both batch and continuous experiments. In the batch experiments, a clear ammonia-LCFA synergetic co-inhibitory effect was identified when the LCFA concentrations were higher than 0.05 g oleate L−1 and ammonia levels between 4.0 and 7.0 NH4+-N L−1. This synergetic effect for LCFA and ammonia levels above 1.1 g oleate L−1 and 4.5 NH4+-N L−1, respectively, was validated in continuous reactors experiments. Nevertheless, adaptation of the AD microbiome to this synergetic co-inhibition could occur after a period of continuous operation. A potential mechanism to explain the synergetic co-inhibition lies on the initial inhibition of methanogens caused by ammonia resulting in increased VFA and hydrogen concentrations, which in turn renders β-oxidation of LCFA thermodynamically unfavourable and thereby brings about further excess accumulation of LCFA and consequently higher unspecific toxicity of all AD steps. This is a vicious cycle, which makes the combined inhibition of the two toxicants more severe, compared to the sum of their individual inhibition effects at the same operational conditions.