Cronobacter sakazakii is an opportunistic pathogen, which causes necrotizing enterocolitis, bacteriaemia and infant meningitis. It has the ability to form biofilm on food contact surfaces, creating food safety risks. In this work, the phenotypic expression of planktonic and biofilm was studied by Fourier transform infrared (FTIR) and Raman spectroscopy. FTIR spectra of the biofilm cells exhibited higher intensity in the absorption bands assigned to polysaccharides, amide I, amide II vibrational mode of ester and carboxylate group. Raman spectra of the biofilm cells showed higher intensity in the absorption band assigned to tyrosine, amide III, carbohydrates, carotenoids, DNA and lipids. Understanding the chemical properties of planktonic and biofilm cells employing the two techniques helped to decipher the differences in the chemical composition between planktonic and biofilm cells. This can promote a better understanding of the persistence, survival and resistance of the biofilm cells.