Understanding how biodiversity changes in time and space is vital to assess the effects of environmental change on benthic ecosystems. Due to the limitations of morphological methods, there has been a rapid expansion in the application of high-throughput sequencing methods to study benthic eukaryotic communities. However, the effect of sample size and small-scale spatial variation on the assessment of benthic eukaryotic diversity is still not well understood. Here, we investigate the effect of different sample volumes in the genetic assessment of benthic metazoan and non-metazoan eukaryotic community composition. Accordingly, DNA was extracted from five different cumulative sediment volumes comprising 100% of the top 2 cm of five benthic sampling cores, and used as template for Ilumina MiSeq sequencing of 18 S rRNA amplicons. Sample volumes strongly impacted diversity metrics for both metazoans and non-metazoan eukaryotes. Beta-diversity of treatments using smaller sample volumes was significantly different from the beta-diversity of the 100% sampled area. Overall our findings indicate that sample volumes of 0.2 g (1% of the sampled area) are insufficient to account for spatial heterogeneity at small spatial scales, and that relatively large percentages of sediment core samples are needed for obtaining robust diversity measurement of both metazoan and non-metazoan eukaryotes.