The biofilms of Staphylococcus aureus on the implanted materials and chronic wounds are life-threatening and are a substantial financial burden on the healthcare system. Silver nanoparticles (SNP), known for their multi-level physiological effects in planktonic cells could be a promising agent in the treatment of biofilm-related infections also. To gain insight into the effects of SNP on various physiological processes in biofilms we studied the transcriptome of Staphylococcus aureus ATCC 29213. To distinguish between ‘nanoparticles-specific’ and ‘ion-specific’ effect of silver, we performed a comparative analysis of the functional genes in response to Ag⁺. As compared to untreated biofilms, 21% (i.e. 629 genes) and 28.5% (i. e. 830 genes) of the total functional coding genes were differentially regulated upon exposure to SNP and Ag⁺. Genes encoding capsular polysaccharides, intercellular adhesion, virulence were downregulated in SNP and Ag⁺ treated biofilms. Genes involved in carbohydrate, protein metabolism including DNA and RNA synthesis, oxidative stress etc. were differentially expressed. Further, activation of efflux pumps and multidrug export proteins was observed, which clearly indicates the presence of metal stress resistance determinants in S. aureus. Silver blocked the integration of mobile genetic elements in S. aureus genome. Our study points out quorum sensing and virulence determinants as possible targets for inhibition of biofilms possibly with/without existing antibiotics. However, further studies on these aspects are warranted. Scanning electron microscopy (SEM) and confocal microscopy revealed changes in biofilm morphology, architecture and thickness in presence of silver nanoparticles and ionic silver, substantiating the transcriptome data.