Diatoms are worldwide used as bioindicators for the assessment of water quality in watercourses. Classically, the diatom biotic indices are based on the relative abundance of morphologically-identified species weighted by their autoecological values. Obtaining such indices is time-consuming, costly and requires excellent taxonomic expertise. Here we tested the possibility to overcome these limitations using a next-generation sequencing (NGS) approach to identify and quantify diatoms found in environmental DNA and RNA samples. We analyzed 27 river sites in Geneva (Switzerland) and compared the values of the Swiss Diatom Index (DI-CH) computed using morphologically-identified species and directly from NGS data. DI-CH showed a significant correlation between morphological and molecular data and similar biological status for the majority of sites. This study demonstrates the potential of the NGS approach for identification and quantification of diatoms in environmental samples, opening new possibilities toward the application of genetic tools for biomonitoring of aquatic ecosystems.