High-throughput growth phenotyping is receiving great attention for establishing the genotype-phenotype map of sequenced organisms owing to the ready availability of complete genome sequences. To date, microbial growth phenotypes have been investigated mostly by the conventional method of batch cultivation using test tubes, Erlenmeyer flasks, or the recently available microwell plates. However, the current batch cultivation methods are time- and labor-intensive and often fail to consider sophisticated environmental changes. The implementation of batch cultures at the nanoliter scale has been difficult because of the quick evaporation of the culture medium inside the reactors. Here, we report a microfluidic system that allows independent cell cultures in evaporation-free multiplex nanoliter reactors under different culture conditions to assess the behavior of cells. The design allows three experimental replicates for each of eight culture environments in a single run. We demonstrate the versatility of the device by performing growth curve experiments with Escherichia coli and microbiological assays of antibiotics against the opportunistic pathogen Pseudomonas aeruginosa. Our study highlights that the microfluidic system can effectively replace the traditional batch culture methods with nanoliter volumes of bacterial cultivations, and it may be therefore promising for high-throughput growth phenotyping as well as for single-cell analyses.