Cronobacter spp. has strong resistance to desiccation and high permeability in Enterobacteriaceae, and powdered infant formula (PIF) is one of the main contamination routes. In recent years, the contamination of Cronobacter spp. in PIF incidents occurs from time to time, causing infant serious diseases or death. In this investigation, matrix-assisted laser desorption/ionization time of flight mass spectrometry was used to identify the phenotypes of 35 Cronobacter strains isolated from PIF and its processing environment. Subsequently, the isolates were evaluated for drying and osmotic pressure tolerance. The results showed that the deactivation rate of the strains ranged from 9.01% to 77.57%, and the highest osmotic pressure condition the strains could tolerate was 6 g/100 mL NaCl. In addition, there was a positive correlation between biofilm formation ability and desiccation resistance. Combined with transcriptomics, Cronobacter spp. could activate biofilm synthesis, produce more trehalose, accumulate betaine and electrolytes to stabilize intracellular structure under the two treatment conditions. A total of 31 and 43 genes were found related to desiccation and permeability resistance, respectively. And some genes (cysM, thuF, ycjO, etc.) were found to be associated with two tolerances for the first time.