Winery effluent is characterized by the presence of high concentrations of organic molecules. Biological sand filters are ideal systems for the bioremediation of this waste at small wineries. In this study, synthetic winery wastewater, consisting of phenolics and high concentrations of ethanol and acetate was treated in experimental biological sand filters operated in batch mode. The organic substrates and metabolites in effluent and pore water samples, taken from four niches (superficial and deep; inlet and outlet), were identified and quantified. The highest COD concentrations were measured at the deep inlet and the lowest at the deep outlet, reflecting the establishment of degradation gradients from inlet to outlet due to plug flow. Ethanol was the preferred substrate in all niches but contrary to expectations, the biodegradation of ethanol and phenolics took place preferentially under lower redox conditions, in the deep niches. There was an accumulation of acetate and propionate, with propionate being found in notably higher ratios in the deep niches. The acidic influent (pH 3.5) was neutralized.