The submission of DNA sequences to public sequence databases is an essential, but insufficiently automated step in the process of generating and disseminating novel DNA sequence data. A user-friendly software tool is needed that streamlines the file preparation for database submissions of DNA sequences that are commonly generated in plant and fungal DNA barcoding. A Python package was developed that converts DNA sequences from the common EMBL and GenBank flat file formats to submission-ready, tab-delimited spreadsheets (so-called "checklists") for a subsequent upload to the annotated sequence section of the European Nucleotide Archive (ENA). The package, titled "EMBL2checklists", automatically converts DNA sequences, their annotation features, and associated metadata into the idiosyncratic format of marker-specific ENA checklists and, thus, generates output that can be uploaded via the interactive Webin submission system of ENA. Here, we present a step-by-step protocol of the bioinformatic steps necessary to generate submission-ready checklist files from raw DNA sequence data and associated metadata via EMBL2checklists.