The recent years have witnessed the capabilities of silver nanoparticles (SNP) as a potent antimicrobial agent. This has resulted in discharge of SNP in the environment and the concentration of particles is steadily increasing. The microbicidal effects of SNP are a serious cause of concern because even low concentrations are likely to cause perturbations in microbial biofilms in aquatic ecosystems causing cascading effects on higher life forms. A clear understanding about ‘particle-specific’ versus ‘ion-specific’ effects on functional genes, particularly in biofilms, is still lacking. We performed a genome-wide transcriptome analysis (via RNA-Seq) of pre-formed P. aeruginosa biofilms in the presence of SNP and Ag⁺ at a concentration of 3.12 μg mL⁻¹. The results of RNA-Seq analysis indicate differential expression of transcripts, 1599 in response to SNP and 2458 for Ag⁺ (i.e. 28 and 43%) as compared to the untreated biofilms. ‘Particle-specific’ effects were evident in the genes involved in biofilm adhesion and dispersion, including vital cellular processes, and genes involved in alkane degradation and denitrification were downregulated. qRT-PCR data on selected genes substantiated our observations. Scanning electron microscopy and confocal microscopy revealed alterations in the cell morphology and a significant reduction in biofilm thickness, indicating dispersion. In medical settings, SNP can eradicate biofilms probably resolving the antibiotic crisis and reducing emerging drug resistance but overuse of particles in consumer products needs regulations so that nutrient cycling, bioremediation, degradation of xenobiotics, etc. are not adversely affected. These results indeed warrant additional studies involving complex microbial communities.