Biofuel cells are a next-generation energy device because they use renewable fuels with high energy density and safety. We have developed passive-type biofuel cell units, which generate a power over 100 mW (80 cm3, 39.7 g). Our biofuel cell, in which two-electron oxidation of glucose and four-electron reduction of O2 occurs at pH 7 in mediated bioelectrochemical processes under quiescent conditions, accomplished the maximum power density of 1.45 ± 0.24 mW cm−2 at 0.3 V. This performance was achieved by introducing three technologies: (1) Enzymes and mediator are densely entrapped on carbon-fiber electrodes with the enzymatic activity retained, (2) the concentration of buffer in electrolyte solution was optimized for the immobilized enzymes, and (3) the cathode structure was designed to supply O2 efficiently. The cell units with a multi-stacked structure successfully operate a radio-controlled car (16.5 g), which demonstrates the potential of biofuel cells in practical applications.