The purpose of this study was to clarify the glass-transition behavior of bacteria (Cronobacter sakazakii) as a function of water activity (aw). From the water sorption isotherm (298 K) for C. sakazakii, monolayer water content and monolayer aw were determined to be 0.0724 g/g-dry matter and 0.252, respectively. Mechanical relaxation was investigated at 298 K. In a higher aw range of over 0.529, the degree of mechanical relaxation increased with an increase in aw. From the effect of aw on the degree of mechanical relaxation, the mechanical awc (aw at which mechanical glass transition occurs at 298 K) was determined to be 0.667. Mean-square displacement of atoms in the bacteria was investigated by incoherent elastic neutron scattering. The mean-square displacement increased gradually with an increase in temperature depending on the aw of samples. From the linear fitting, two or three dynamical transition temperatures (low, middle, and high Tds) were determined at each aw. The low-Td values (142–158 K) were almost independent from aw. There was a minor effect of aw on the middle Td (214–234 K) except for the anhydrous sample (261 K). The high Td (252–322 K) largely increased with the decrease in aw. From the aw dependence of the high Td, the dynamical awc was determined to be 0.675, which was almost equivalent to the mechanical awc. The high Td was assumed to be the glass-transition temperature (Tg), and anhydrous Tg was estimated to be 409 K. In addition, molecular relaxation time (τ) of the bacteria was calculated as a function of aw. From the result, it is suggested that the progress of metabolism in the bacterial system requires a lower τ than approximately 6 × 10−5 s.