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 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 nanoliter droplet of cell suspension encapsulated by a polydimethylsiloxane (PDMS) membrane. By manipulating the chemistry of their polymeric shell, the nanocultures can achieve functionalities, such as selective permeability facilitating the transport of metabolites and other small molecules essential to cell growth and community dynamics. The nanocultures allow the diffusion of antibiotics, signaling molecules, and functional fluorescent probes to interrogate cell physiology and facilitate microbial interactions across the confining vessel. Alternatively, multiple species of microbes can be co-cultured within the nanocultures. Because of the chemical exchange occurring within and across the shell, the nanocultures can help investigate microbial pathophysiology. Here, we demonstrate this platform by exploring broad ranges of direct and indirect microbial dynamics. This versatile new tool has broad potential for addressing biological questions associated with drug resistance, chronic infections, antibiotic discovery, and microbiome dynamics relevant to complex microbial communities.