RNA-sequencing for whole transcriptome analysis requires high-quality RNA in adequate amounts, which can be difficult to generate with low-biomass-producing bacteria where sample volume is limited. We present an RNA extraction protocol for low-biomass-producing autotrophic bacteria Nitrosomonas europaea and Nitrobacter winogradskyi cultures. We describe steps for sample collection, lysozyme-based enzymatic lysis, and a commercial silica-column-based RNA extraction. We then detail evaluation of RNA yield and quality for downstream applications such as RNA-Seq. For complete details on the use and execution of this protocol, please refer to Verbeelen et al.1.