Mucosal and barrier tissues such as the gut, lung or skin, are composed of a complex network of cells and microbes forming a tight niche that prevents pathogen colonization and supports host-microbiome symbiosis. Characterizing these networks at high molecular and cellular resolution is crucial for our understanding of homeostasis and disease. Spatial transcriptomics has emerged as a key technology to positionally profile RNAs at high resolution in tissues. Here, we present spatial host-microbiome sequencing, an all-sequencing based approach that captures tissue histology, polyadenylated RNAs and bacterial 16S sequences directly from tissues on spatially barcoded glass surfaces. We apply our approach to the mouse gut as a model system, use a novel deep learning approach for data mapping and detect spatial niches impacted by microbial biogeography. Spatial host-microbiome sequencing should enhance study of native host-microbe interactions in health and disease.