Background Phylogenetic trees are an important analytical tool for examining species and community diversity, and the evolutionary history of species. In the case of microorganisms, decreasing sequencing costs have enabled researchers to generate ever-larger sequence datasets, which in turn have begun to fill gaps in the evolutionary history of microbial groups. However, phylogenetic analyses of large sequence datasets present challenges to extracting meaningful trends from complex trees. Scientific inferences made by visual inspection of phylogenetic trees can be simplified and enhanced by customizing various parts of the tree, including label color, branch color, and other features. Yet, manual customization is time-consuming and error prone, and programs designed to assist in batch tree customization often require programming experience. To address these limitations, we developed Iroki, a program for fast, automatic customization of phylogenetic trees. Iroki allows the user to incorporate information on a broad range of metadata for each experimental unit represented in the tree. Results Iroki was applied to four existing microbial sequence datasets to demonstrate its utility in data exploration and presentation. Specifically, we used Iroki to highlight connections between viral phylogeny and host taxonomy, explore the abundance of microbial groups associated with Shiga toxin-producing Escherichia coli (STEC) in cattle, examine short-term temporal dynamics of virioplankton communities, and to search for trends in the biogeography of Zetaproteobacteria. Conclusions Iroki is an easy-to-use application having both command line and web-browser implementations for fast, automatic customization of phylogenetic trees based on user-provided categorical or continuous metadata. Iroki enables hypothesis testing through improved visualization of phylogenetic trees, streamlining the process of biological sequence data exploration and presentation. Availability Iroki can be accessed through a web browser application or via installation through RubyGems, from source, or through the Iroki Docker image. All source code and documentation is available under the GPLv3 license at https://github.com/mooreryan/iroki. The Iroki web-app is accessible at www.iroki.net or through the VIROME portal (http://virome.dbi.udel.edu), and its source code is released under GPLv3 license at https://github.com/mooreryan/iroki_web. The Docker image can be found here: https://hub.docker.com/r/mooreryan/iroki.