A compartmentalized microfluidic chamber array that offers not only separate cell culture environments but also independent control of the diffusion of small molecules provides an extremely useful platform for cell cultivations and versatile cellular assays. However, it is challenging to incorporate both cell compartmentalization and active diffusion control in real-time and precise manners. Here, we present a novel nanoscale hydrodynamic film (NHF) that is formed between a solid substrate and a polydimethylsiloxane (PDMS) surface. The thickness of the NHF can be adjusted by varying the pressure applied between them so that the mass transfer through the NHF can also be controlled. These novel phenomena are characterized and applied to develop a compartmentalized microchamber array with diffusion-tunable and solution-switchable chemostat-like versatile bacterial assays. The NHF-based compartmentalization technique is ideal for not only continuous bacterial cultivation by consistently refreshing various nutrient sources but also various diffusion-based microbial assays such as chemical induction of synthetically engineered bacterial cells and selective growth of a specific bacterial strain with respect to chemical environments. In addition, we show that tight compartmentalization protects cells in the chambers, while biofilm formation and nutrient contamination are eliminated by loading a lysis buffer, which typically hinders long-term continuous cultures and accurate microbial assays on a chip. Therefore, we ensure that the NHF-based compartmentalization platform proposed in this work will facilitate not only fundamental studies in microbiology but also various practical applications of microbes for production of valuable metabolites and byproducts in a high-throughput and highly efficient format.