Bacterial biofilms are microbial lifestyles found in all environments . Up to 80% of human infections and 60–70% of hospital-acquired infections have a biofilm origin, with Staphylococcus aureus one of the leading causes of these infections . Microorganisms in biofilms exhibit significant antimicrobial resistance which poses important treatment challenges, hence the urgent need to identify novel antibiofilm strategies . Microbes form biofilms in response to various factors, and once these 3-dimentional structures form they are highly recalcitrant to removal . The switch from planktonic lifestyle to the biofilm protected mode of growth results in a phenotypic shift in the behavior of the microorganisms in terms of growth rate and gene expression . Given these changes, investigation of microbial gene expression and their modulation at different stages of biofilm maturation is needed to provide vital insight into the behavior of biofilm cells . In this study, we analyzed publicly available transcriptomic dataset of S. aureus biofilms at different stages of maturation to identify consistently upregulated genes irrespective of the biofilm maturation stage . Our reanalysis identified a total of 6 differentially expressed genes upregulated in both 48 and 144-h old S. aureus biofilms . Functional analysis revealed that these genes encode for proteins which play a role in key microbial metabolic pathways . However, these genes, as yet, are unrelated or fully studied in the context of biofilm . Moreover, the findings of this in silico work, suggest that these genes may represent potential novel targets for the development of more effective antibiofilm strategies against S. aureus biofilm-associated infections.