This data set concerns the biochemical and biophysical characterization of three lactic acid bacteria exhibiting different resistance to freezing to identify markers of cryoresistance. Carnobacterium maltaromaticum CNCM I-3298 (most freeze-resistant), Lactobacillus delbrueckii ssp bulgaricus ATCC 11842 and Lactobacillus delbrueckii ssp bulgaricus CFL1 (most freeze-sensitive) were investigated. The dataset includes for each of the three strains: i) biological activity (culturability and acidifying activity) and ii) biochemical composition by Fourier transform infrared microscopy of cells in an aqueous environment before and after freeze-thawing; iii) membrane characterization: fatty acid composition, membrane fluidity following cooling, lipid phase transition following freezing and iv)intracellular glass transition temperature