The chemostat is an effective method for bacterial studies that require monitoring and manipulation of proliferation/growth in response to physicochemical stimulation. Despite of its effectiveness, its potentially possible applications were limited due to current challenges in achieving continuous growth and population control simultaneously. Here, we present a microchemostat that enables reversible bacterial isolation and continuous chemical refreshment using mass transport-tunable nanoscale hydraulic films (NHF). Because the microchemostat controls bacterial growth and subculture conditions and operates in a fully automated and programmed manner, it is possible to manipulate bacterial populations for long-term with ultra-low reagent consumption. For demonstrations, the proposed microchemostat could be used in bacterial studies including genetic induction and antibiotic resistance and practical applications in microbial biosensors. We envision that our microchemostat is expected to be applied to much wider range of microbial studies or applications on a single chip.