The present study aimed to investigate the response of macrobenthic assemblages along a gradient of metal contamination using a combination of uni- and multivariate methods focusing on their composition, structure and function. A total of six sites were established based on a preliminary survey, which identified three areas with different levels of contamination. These areas were defined as slightly contaminated (SC), moderately contaminated (MC) and highly contaminated (HC). Each area comprised two sites, sampled in four sampling surveys (September 2012, February, May and October of 2013). To investigate the response of the macrobenthic assemblages the number of individuals (N), number of taxa (S), Shannon-Weaver diversity (H'), Pielou's equitability (J') and different distance-based multivariate measures of β-diversity (complementarity) were analysed. β-diversity as turnover was also analysed together with spatial and temporal changes in the trophic structure. A clear gradient of increasing contamination was consistently detected, but comparisons with available sediment quality guidelines indicated that adverse biological effects may be expected in all areas. This result suggests measuring concentrations of contaminants in the sediment per se may be insufficient to establish a clear link between ecological patterns and the contamination of the system. Also it highlights the difficulty of identifying reference areas in highly urbanized and industrialized estuaries. Only multivariate analysis (dbRDA; both using the taxonomic and trophic composition) and β-diversity as turnover showed a consistent response to metal contamination. Higher heterogeneity, mainly due to contribution of rare species (i.e. species present in a single sampling period), was observed in the least contaminated area (SC), decreasing towards the HC. In terms of the trophic function, a shift from a dominance of carnivores in the SC to the dominance of deposit-feeding organisms (and associations) along the contamination gradient was evident. © 2015 Elsevier Ltd.