Water sorption isotherms of bacteria reflect the water activity with the change of moisture content of bacteria at a specific temperature. The temperature-dependency of water activity change can help to understand the thermal resistance of bacteria during a thermal process. Thermal resistance of bacteria in low-moisture foods may differ significantly depending on the physiological characteristics of microorganisms, including cell structure, existence of biofilms, and growth state. Previous studies demonstrated that the incremental change of aw in bacterial cells during thermal treatments resulted in changes in their thermotolerance. In this study, a pathogen associated with low-moisture foods outbreaks, Salmonella Enteritidis PT30 (in planktonic and biofilm forms), and its validated surrogate, Enterococcus faecium, were lyophilized and their water sorption isotherms (WSI) at 20, 40, and 60 °C were determined by using a vapor sorption analyzer and simulated by the Guggenheim, Anderson and De Boer model (GAB). The published thermal death times at 80 °C (D80 °C-values) of these bacteria in low-moisture environments were related with their WSI-derived aw changes. The results showed that planktonic E. faecium and biofilms of Salmonella, exhibiting higher thermal resistance compared to the planktonic cultures of Salmonella, had a smaller increase in aw when thermally treated from 20 to 60 °C in sealed test cells. The computational modeling also showed that when temperature increased from 20 to 60 °C, with an increase in relative humidity from 10% to 60%, freeze-dried planktonic E. faecium and Salmonella cells would equilibrate to their surrounding environments in 0.15 s and 0.25 s, respectively, suggesting a rapid equilibration of bacterial cells to their microenvironment. However, control of bacteria with different cell structure and growth state would require further attentions on process design adjustment because of their different water sorption characteristics.