The analysis of benthic assemblages is a valuable tool to describe the ecological status of transitional water ecosystems, but species are extremely sensitive and respond to both microhabitat and seasonal differences. The identification of changes in the composition of the macrobenthic community in specific microhabitats can then be used as an “early warning” for environmental changes which may affect the economic and ecological importance of lagoons, through their provision of Ecosystem Services. From a conservational point of view, the appropriate definition of the spatial aggregation level of microhabitats or local communities is of crucial importance. The main objective of this work is to assess the role of the spatial scale in the analysis of lagoon biodiversity. First, we analyze the variation in the sample coverage for alternative aggregations of the monitoring stations in three lagoons of the Po River Delta. Then, we analyze the variation of a class of entropy indices by mixed effects models, properly accounting for the fixed effects of biotic and abiotic factors and random effects ruled by nested sources of variability corresponding to alternative definitions of local communities. Finally, we address biodiversity partitioning by a generalized diversity measure, namely the Tsallis entropy, and for alternative definitions of the local communities. The main results obtained by the proposed statistical protocol are presented, discussed and framed in the ecological context.