Abstract Motivation The submission of annotated sequence data to public sequence databases constitutes a central pillar in biological research. The surge of novel DNA sequences awaiting database submission due to the application of next-generation sequencing has increased the need for software tools that facilitate bulk submissions. This need has yet to be met with the concurrent development of tools to automate the preparatory work preceding such submissions. Results The author introduce annonex2embl, a Python package that automates the preparation of complete sequence flatfiles for large-scale sequence submissions to the European Nucleotide Archive. The tool enables the conversion of DNA sequence alignments that are co-supplied with sequence annotations and metadata to submission-ready flatfiles. Among other features, the software automatically accounts for length differences among the input sequences while maintaining correct annotations, automatically interlaces metadata to each record and displays a design suitable for easy integration into bioinformatic workflows. As proof of its utility, annonex2embl is employed in preparing a dataset of more than 1500 fungal DNA sequences for database submission. Availability and implementation annonex2embl is freely available via the Python package index at http://pypi.python.org/pypi/annonex2embl. Supplementary information Supplementary data are available at Bioinformatics online.