Benthic foraminiferal assemblages are employed for past environmental reconstructions, as well as for biomonitoring studies in recent environments. Despite their established status for such applications, and existing protocols for sample treatment, not all studies using benthic Foraminifera employ the same methodology. For instance, there is no broad practical consensus whether to use the >125 or >150 μm size fraction for benthic foraminiferal assemblage analyses. Here, we use early Pleistocene material from the Pefka E section on the Island of Rhodes (Greece), which has been counted in both size fractions, to investigate whether a 25 μm difference in the counted fraction is already sufficient to have an impact on ecological studies. We analyzed the influence of the difference in size fraction on studies of biodiversity as well as multivariate assemblage analyses of the sample material. We found that for both types of studies, the general trends remain the same regardless of the chosen size fraction, but in detail significant differences emerge which are not consistently distributed between samples. Studies which require a high degree of precision can thus not compare results from analyses that used different size fractions, and the inconsistent distribution of differences makes it impossible to develop corrections for this issue. We therefore advocate the consistent use of the >125 μm size fraction for benthic foraminiferal studies in the future.