Diatom identification and counting by light microscopy of permanently embedded acid-cleaned silicate shells (frustules) is a fundamental method in ecological and water quality investigations. Here we present a new variant of this method based on “digital virtual slides”, and compare it to the traditional, non-digitized light microscopy workflow on freshwater samples. We analysed three replicate slides taken from six benthic samples using two methods: (1) working directly on a light microscope (the “traditional” counting method), and (2) preparing “virtual digital slides” by high-resolution slide scanning and subsequently identifying and labelling individual valves or frustules using a web browser-based image annotation platform (the digital method). Both methods led to comparable results in terms of species richness, diatom indices and diatom community composition. Although counting by digital microscopy was slightly more time consuming, our experience points out that the digital workflow can not only improve the transparency and reusability of diatom counts but it can also increase taxonomic precision. The introduced digital workflow can also be applied for taxonomic inter-expert calibration through the web, and for producing training image sets for deep-learning-based diatom identification, making it a promising and versatile alternative or extension to traditional light microscopic diatom analyses in the future.