Microbe-microbe and host–microbe interactions are central to the functioning of the human microbiome, but few tools are available to measure these interactions spatially within tissues. In this talk, I will introduce multiplexed imaging and spatial RNA sequencing approaches that enable simultaneous mapping of gut microbial communities, host gene expression and host gene regulation at single-micron resolution. By applying these methods, we reveal the longitudinal stability of spatial architectures in the healthy human oral plaque microbiome, demonstrate the location-dependent organization of microbial communities in the mouse intestine, and reveal interactions within and between microbial taxa at short length scales. I will also show how these tools reveal changes in microbial and host cell architectures at microbiome–tumor interfaces. Last, I will discuss recent extensions of these methods to enable spatial mapping of mobile genetic elements and their bacterial hosts in complex microbiomes. Together, these methods provide a new avenue to study host-microbiome interactions, with the potential for advances in microbiome science.