We demonstrated a size sieving microbial cell separation apparatus consisting of multipillar microenclosures. The apparatus consists of two kinds of microenclosures that differ in size, namely, 4 × 4 µm2 enclosures with 0.2 µm space between 1 µm diameter pillars and 8 × 8 µm2 enclosures with 1 µm space. Single cells of Escherichia coli and Saccharomyces cerevisiae were separately captured in smaller and larger microenclosures, respectively, just by dropping mixtures. Moreover, we examined whether incubation using the microenclosure structure was possible or not, and succeeded in the incubation of S. cerevisiae. Microlens arrays using prefabricated mesa structures by the Ar plasma etching process were also manufactured. We believe that the fluorescence analysis or investigation of photochemical reactions for microbial single cells can be carried out by a combination of the microenclosure structure and optical devices.