The effects of winter flooding on the intertidal macrobenthic assemblages in the Guadiana estuary were studied by analysing several ecological parameters (abundance, biomass, species richness, diversity, degree of dominance of annelids and deposit-feeders species and number of predator species) as well as changes in the composition of the assemblages. Significant and systematic differences were found in the values of the ecological parameters, especially between winter and summer. We found a significant interaction between the factors ‘time’ and ‘site’, attributable to changes in abundance and composition of the assemblages. The assemblages showed a predominance of some opportunist species in most of the estuary after the flooding, suggesting that these species benefited from the new environmental conditions. Conversely, the assemblages in the mouth of the estuary were severely affected as was shown by the negative changes in biomass, species richness and diversity. These converse responses of the intertidal estuarine assemblages represent effects of flooding different from both catastrophic and negative effects on macrofaunal assemblages.