The importance of sampling effort in the statistical exploration of spatial autocorrelation is demonstrated for benthic macroinvertebrate assemblages within the intertidal warm-temperate Knysna estuary, South Africa. While the role of spatial scale in determining autocorrelation patterns in ecological populations has been noted, the effects of changing sampling effort (e.g., sample size) have rarely been explored; neither have the nature of any changes with sample size. Invertebrate assemblages were sampled from a single grid lattice comprised of 48 sampling stations at four sample sizes (0.0015, 0.0026, 0.0054 and 0.01 m 2 ). Four metrics were investigated: assemblage abundance, frequency (species density), and numbers of the two most abundant species in the area Simplisetia erythraeensis and Prionospio sexoculata. Spatial autocorrelation was estimated for each sample size from the global Moran’s I. For a range of distance classes, Moran’s I correlograms were constructed, these plotted autocorrelation estimates as a function of the separation distance between point samples. Spatial autocorrelation was present in three of the metrics (assemblage abundance frequency and Prionospio abundance), but not for Simplisetia abundance. The estimated magnitude of spatial autocorrelation varied across sampling units for all four assemblage and species metrics (global Moran’s I ranged from 0.5 to − 0.07). Correlograms indicated that optimal sampling interval distances fell in the region of 8 m for Simplisetia and 19 m for the remaining three metrics. These distances indicate the dimensions of the processes (both biotic and abiotic) that determine spatial patterning in the microbenthic intertidal invertebrates sampled.