This paper deals with the quantitative measurement of the influence of different inhibitory compounds present in olive oil mill and winery industrial wastewaters on their anaerobic digestion treatment. Specifically, the physical separation of the two stages (acidogenic and methanogenic) that comprise the anaerobic digestion process, and the acclimation time of the microorganisms have been evaluated. First, a series of experiments with glucose model solutions made clear that external pH control (>8) in methanogenic reactor is a key factor to guarantee suitable overall chemical oxygen demand (COD) removals and biogas production rates in a two-phase operation. Next, a response surface methodology (RSM) was applied to quantify the effect of olive oil, ethanol and phenol (in form of synthetic solutions simulating diluted real wastewaters) on the two above mentioned responses. According to the results of this method, inhibiting power of these compounds follows the order: ethanol >> phenol olive oil. Although the general trends observed in a two-stage anaerobic digestion process do not differ much from the ones obtained in a single-stage process, it should be emphasized the higher stability of the process, with yields >60% even in the presence of moderate concentrations of inhibiting compounds (0.25% w/w olive oil, 75 mg L-1 phenol and 125 mg L-1 ethanol). (C) 2012 Elsevier B.V. All rights reserved.