AbstractStaphylococcus aureusis a major opportunistic pathogen that commonly forms biofilms on various biotic and abiotic surfaces. Also, most isolates are known to carry prophages in their genomes. With this in mind, it seems that acquiring a better knowledge of the impact of prophages on the physiology ofS.aureusbiofilm cells would be useful for developing strategies to eliminate this pathogen. Here, we performed RNA-seq analysis of biofilm cells formed byS.aureusRN450 and two derived strains carrying prophages ϕ11 and ϕ80α. The lysogenic strains displayed increased biofilm formation and production of the carotenoid pigment staphyloxanthin. These phenotypes could be partly explained by the differences in gene expression displayed by prophage-harboring strains, namely an activation of the alternative sigma factor (SigB) regulon and downregulation of genes controlled by the Agr quorum-sensing system, especially the decreased transcription of genes encoding dispersion factors like proteases. Nonetheless, spontaneous lysis of part of the population could also contribute to the increased attached biomass. Interestingly, it appears that the phage CI protein plays a role in orchestrating these phage-host interactions, although more research is needed to confirm this possibility. Likewise, future studies should examine the impact of these two prophages during the infection.