Abstract The integration of many disparate forms of data is essential for understanding the microbial world and its interaction with the environment and human health. Doing so is particularly challenging in the context of microbe-host and microbe-microbe interactions that contribute to health or environmental outcomes. There are often thousands of relevant microbial species, and millions of interactions among those microbes and with their environment or host. Some experimental observations only distinguish coarser taxonomic resolutions such as family or phylum-level. Integrated information (e.g., about host and microbial physiology, genetics, and metabolism) facilitates deeper understanding of complex interactions and helps interpret correlative results. The KG-Microbe construction framework is a novel approach to harmonizing bacterial and archaeal data in the form of a knowledge graph (KG). Starting from a core KG with organismal traits, environments and growth preferences, the framework generates a hierarchy of related KGs targeting specific conceptual use cases, including the human host-associated microbiome in the context of disease. KG-Microbe is a standardized and interoperable framework that integrates microbial organismal and genomic traits, represented ontologically, for biomedical, environmental, and other applications. The framework supports customizable taxa subsets representing microbial lineages or communities of interest. Evaluations of the KG-Microbe knowledge graphs through a series of competency questions demonstrate the accuracy and effectiveness of the data harmonization, and the utility of the resulting KGs in inflammatory bowel and Parkinson’s diseases. Finally, the predictive and environmental capabilities of the KGs are demonstrated by explaining growth preferences through training a model using graph features. KG-Microbe is a flexible, modular enabling technology for humans and machine learning methods to uncover mechanistic explanations of microbial associations.