Psychrotophic Pseudomonas species, and in particular Pseudomonas fragi and Pseudomonas lundensis, dominate the spoilage of chilled, aerobically stored red meat. While they are known to be the cause of “slime” on the meat, which is regarded as a major quality defect, little is known about the characteristics of this biofilm. To study biofilm formation by these bacteria in situ a model system that closely mimics the practical industry conditions under which raw beef muscle is stored was developed. Using this system, biofilm structure using confocal laser scanning microscopy, biofilm matrix composition using chemical analysis and Raman spectroscopy, and biofilm gene expression using RNA sequencing were investigated. Structural differences between the biofilms formed by P. fragi and P. lundensis were apparent. In particular, P. fragi biofilms were characterised by very dense and compact cells with nemetic ordering, while P. lundesis biofilms were more loosely arranged with voids. The content of protein and carbohydrate in the matrix of these bacteria was found to be higher when they were grown at lower temperatures. Genes encoding Flp family type IVb pilin, ribosome modulation factor, creatininase and pyruvate dehydrogenase were upregulated during biofilm maturation while genes encoding for iron uptake systems and taurine transport were significantly down regulated. Protein synthesis was shown to cease once the biofilm had reached its maximum density. This study provided significant insights in biofilm formation by these bacteria and new potential targets to control their proliferation and extend the shelf-life of aerobically stored red meat.