Abstract: The need for assessment tools for microbial dynamics has necessitated the miniaturization of cell-culturing techniques, and the design of microsystems that facilitate the interrogation of microorganisms in-well-defined environments. The nanocultures, as described in this work, are such an assessment tool: nanoliter-sized microcapsules generated using a flow-focusing microfluidic device to sequester and cultivate microbes in a high-throughput manner. Each nanoculture begins as a several nanoliter droplets of suspended cells, encapsulated by a polydimethylsiloxane (PDMS) membrane. By manipulating the chemistry of their polymeric shell, the nanocultures can be designed to achieve functionalities, such as selective permeability facilitating the transport of metabolites and other small molecules essential to control cell growth and characterize community dynamics. The nanocultures allow the diffusion of functional probes to interrogate cell physiology under chemical insults, allowing microbial interactions to be probed within or across the confining vessel. Alternatively, multiple species of microbes can be co-cultured within a nanoculture. Because chemical communication across the membrane occurs, this system can be used to decouple the effects of the physicochemical interactions between cells and investigate microbial pathophysiology. Overall, our work paves the way in our understanding of effectively using the nanocultures to study complex synergistic and antagonistic microbial behaviors in both natural and synthetic communities, with the goal of better simulating natural microenvironments and increasing discoverability of unknown molecules that are relevant to complex microbial communities.