Molecular mechanisms underlying the thermal response of cells remain elusive. On the basis of the recent result that the short-time diffusive dynamics of the Escherichia coli proteome is an excellent indicator of temperature-dependent bacterial metabolism and death, we used neutron scattering (NS) spectroscopy and molecular dynamics (MD) simulations to investigate the sub-nanosecond proteome mobility in psychro-, meso-, and hyperthermophilic bacteria over a wide temperature range. The magnitude of thermal fluctuations, measured by atomic mean square displacements, is similar among all studied bacteria at their respective thermal cell death. Global roto-translational motions turn out to be the main factor distinguishing the bacterial dynamical properties. We ascribe this behavior to the difference in the average proteome net charge, which becomes less negative for increasing bacterial thermal stability. We propose that the chemical–physical properties of the cytoplasm and the global dynamics of the resulting proteome are fine-tuned by evolution to uphold optimal thermal stability conditions.