Research in marine protected areas (MPAs) needs to focus beyond targeted species to the functional value of MPAs in maintaining ecosystem services and ecological diversity. Estuarine tidal flats are speciose and provide vital ecosystem services but are largely neglected in MPA research. Here, the ecological effect of an MPA on an estuarine tidal flat was determined by quantifying patterns in macroinvertebrate assemblages and sediment variables over a 3 yr period: 1 yr prior to and 2 yr following MPA zoning. An asymmetrical beyond BACI (before after control impact) design was used with 1 protected and 2 reference tidal flats. Following the exclusion of humans targeting callianasid crustaceans for bait, significant changes in the assemblages were observed in the no-take zone compared to reference locations. These shifts were maintained for the 2 yr of sampling following zoning. Relatively immobile, suspension- and deposit-feeding species increased up to 6-fold in abundance, especially juvenile bivalves (e.g. Eumarcia fumigata and Soletellina alba) and small polychaetes. In contrast, there was a reduction in some of the highly mobile, predatory and scavenging species (e.g. the amphipod Urohaustorius metungi and the polychaete Sigalion ovigerum). We observed an increase in spatial homogeneity in the assemblage as well as increases in the silt and clay content at the protected flat, while patchiness was maintained at the reference sites. Importantly, these results add to the growing body of evidence that MPAs also significantly affect non-target fauna and produce shifts in beta-diversity. Our findings imply that MPAs are an effective tool for conservation management.