Abstract Background: It is known that aquaculture may produce negative environmental effects on marine ecosystems. Southern Chile is one of the most important salmon and mussel-producing areas in the world. Here we assess the ecological status of benthic communities near farming centers in Caucahue Channel, Chiloe, which has been used intensely for salmon and mussel production for 30 years. Methods: The macrofauna, sediments and water column were characterized at distances of 5 to 100 m from three salmon and three mussel-producing centers. Information was also obtained from reference sites 500 to 3000 m from these aquaculture farms. The macrofauna and environmental conditions during winter were analyzed using uni- and multivariate analysis and the AZTI Marine Biotic Index (AMBI) as an indicator of benthic community condition. Results: (i) There is a high degree of spatial dissimilarity in macrofauna and environmental variables among sampling sites and types of environments (far from or near farming centers) and between the northern and southern areas of the channel; (ii) sediment structure (mean grain size and percentage of total organic matter) correlated with the observed dissimilarities in macrofauna communities; and (iii) the level of perturbation according to AMBI was heterogeneous, with sites in the undisturbed/normal range to moderately disturbed/polluted. Conclusions: We found a high spatial dissimilarity in benthic macrofauna and environmental variables among sampling sites, environmental types and between the northern and southern areas of the channel. AMBI and multivariate community-environment analysis are useful tools to define the level of perturbation of a geographic area at different spatial scales, using all the ecological information from each sample and replicates.