Benthic macrofauna is regularly used in monitoring programmes, however the vast majority of benthic eukaryotic biodiversity lies mostly in microscopic organisms, such as meiofauna (invertebrates < 1 mm) and protists, that rapidly responds to environmental change. These communities have traditionally been hard to sample and handle in the laboratory, but DNA sequencing has made such work less time consuming. Compared to DNA sequencing that captures both alive and dead organisms, environmental RNA (eRNA) can be used to better target alive communities. Here, we assessed the biodiversity of three known bioindicator microeukaryote groups (nematodes, foraminifera, and ciliates) in sediment samples collected at seven coastal sites along an organic carbon (OC) gradient. We aimed to investigate if eRNA shotgun sequencing can be used to simultaneously detect differences in 1) biodiversity of multiple microeukaryotic communities, and 2) functional feeding traits of nematodes. Results showed that biodiversity was lower for nematodes and foraminifera in high OC (6.2–6.9 %), when compared to low OC sediments (1.2–2.8 %). The beta diversity for all three groups were different along the OC gradient, as well as the classified feeding type of nematode genera (with more non-selective deposit feeders in high OC sediment). High relative abundant genera included nematode Sabatieria and foraminifera Elphidium in high OC, and Cryptocaryon-like ciliates in low OC sediments. Considering that future sequencing technologies are likely to decrease in cost, the use of eRNA shotgun sequencing to assess biodiversity of living benthic microeukaryotes could be a powerful tool in recurring monitoring programmes.