Biofouling is a major challenge in the water industry and public health. Silver nanoparticles (AgNPs) have excellent antimicrobial properties and are considered to be a promising anti-biofouling agent. A modified method was used to produce small sized and well-dispersed biogenic silver nanoparticles with a mean size of ~6 nm (Bio-Ag0-6) using Lactobacillus fermentum. The morphology, size distribution, zeta potential and oxidation state of the silver were systematically characterized. Determination of minimal inhibitory and bactericidal concentration results revealed that biogenic silver Bio-Ag(0-6) can effectively suppress the growth of the test bacteria. Additionally, the inhibition effects of Bio-Ag(0-6) on biofilm formation and on established biofilms were evaluated using P. aeruginosa (ATCC 27853) as the model bacterium. The results from microtiter plates and confocal laser scanning microscopy demonstrated that Bio-Ag(0-6) not only exhibited excellent antibacterial performance but also could control biofilm formation and induce detachment of the bulk of P. aeruginosa biofilms leaving a small residual matrix.