Abstract Accurate and accessible phylogenetic analysis is essential for understanding microbial taxonomy and evolution, which are integral to microbiology, ecology, and drug discovery, yet it remains a challenging task. AutoMLST2 (https://automlst2.ziemertlab.com) is a web server designed to facilitate automated phylogenetic reconstruction and microbial taxonomy analysis for bacterial and archaeal genomes. It builds on the foundation of AutoMLST, which remains widely used due to its user-friendly interface compared to similar tools. Given its continued popularity and utility, we have enhanced AutoMLST to leverage newer reference databases and computational tools. AutoMLST2 integrates the Genome Taxonomy Database, extends support to archaeal genomes, and improves analytical flexibility. Key improvements include more customizable processing modes, containerization to prevent queue accumulations, and parallel computing for large-scale studies. By incorporating up-to-date databases and workflows, AutoMLST2 continues to provide an accessible and efficient platform for researchers in microbiology, evolutionary ecology, and natural product discovery.