The influx of whole genome sequencing (WGS) data in the public health and clinical diagnostic sectors has created a need for data analysis methods and bioinformatics expertise, which can be a bottleneck for many laboratories. At Sciensano, the Belgian national public health institute, an intuitive and user-friendly bioinformatics tool portal was implemented using Galaxy, an open-source platform for data analysis and workflow creation. The Galaxy @Sciensano instance is available to both internal and external scientists and offers a wide range of tools provided by the community, complemented by over 50 custom tools and pipelines developed in-house. The tool selection is currently focused primarily on the analysis of WGS data generated using Illumina sequencing for microbial pathogen typing, characterization and outbreak detection, but it also addresses specific use cases for other data types. Our Galaxy instance includes several custom-developed ‘push-button’ pipelines, which are user-friendly and intuitive stand-alone tools that perform complete characterization of bacterial isolates based on WGS data and generate interactive HTML output reports with key findings. These pipelines include quality control, de novo assembly, sequence typing, antimicrobial resistance prediction and several relevant species-specific assays. They are tailored for pathogens with active genomic surveillance programs, and clinical relevance, such as Escherichia coli, Listeria monocytogenes, Salmonella spp. and Mycobacterium tuberculosis. These tools and pipelines utilize internationally recognized databases such as PubMLST, EnteroBase, and the NCBI National Database of Antibiotic Resistant Organisms, which are automatically synchronized on a regular basis to ensure up-to-date results. Many of these pipelines are part of the routine activities of Belgian national reference centers and laboratories, some of which use them under ISO accreditation. This resource is publicly available for noncommercial use at https://galaxy.sciensano.be/ and can help other laboratories establish reliable, traceable and reproducible bioinformatics analyses for pathogens encountered in public health settings.