Psychrotrophic Pseudomonas species are the key spoilage bacteria of aerobically stored chilled meat. These organisms readily form biofilms on meat under refrigerated conditions leading to consumer rejection and associated economic losses. Limited information is available on the matrix composition of the biofilms formed by these bacteria. We quantified and characterized the main components of the matrix of mono-species biofilms of selected Pseudomonas fragi and Pseudomonas lundensis strains using chemical analysis and Raman spectroscopy. The biofilms were grown at 10 °C and 25 °C on nitro-cellulose membranes placed on surface sterilized beef cuts. Extra-cellular polymeric substances of the matrix were extracted in soluble and bound forms and were chemically assessed for total carbohydrates, proteins and extra-cellular DNA. Both Pseudomonas species showed a significant increase in total carbohydrates and total proteins when grown at 10 °C as compared to 25 °C. Extra-cellular DNA did not show a strong correlation with growth temperature. Raman spectra were obtained from planktonic bacteria and membrane grown biofilms at 10 °C and 25 °C. Higher levels of guanine were detected in planktonic cells as compared to biofilm cells. This study suggests that psychrotrophic Pseudomonas species may respond to cold stress by increasing extra-cellular polymer secretions.