In this paper we demonstrate highly dense immobilization of bacteria into nanoimprinted holes. Nanoimprinting enables micro holes smaller than 2 μm in diameter with a high accuracy, which cannot be patterned using conventional UV photolithography. In our prior work, we developed a microbial reactor immobilizing bacteria into micro holes, which facilitated collection and evaluation of reaction products while the number of bacteria involved in the reaction could be quantified. However, the holes were made by photolithography and the minimum size was limited to be 3 μm in diameter. Large holes allow multiple bacteria to be immobilized in a hole, which resulted in errors in quantification. The number of bacteria immobilized in a nanoimprinted hole was found to have smaller deviation than in photolithographically formed holes. In addition, density of the immobilized bacteria was experimentally found to be largest in case of 2-μm-holes. The proposed processes will be of great help for precise evaluation of bacteria reaction.