Pseudomonads produce several lipopeptide biosurfactants that have antimicrobial properties, but also facilitate surface motility and influence biofilm formation. Detailed studies addressing the significance of lipopeptides for biofilm formation and architecture are rare. Hence the current study sets out to determine the specific role of the lipopeptide viscosin for Pseudomonas fluorescens SBW25 biofilm formation, architecture and dispersal, and to relate viscA gene expression with viscosin production and effect. Initially, we compared biofilm formation of SBW25 and the viscosin-deficient mutant strain SBW25ΔviscA in static microtiter assays. These experiments demonstrated that viscosin had little influence on the amount of biofilm formed by SBW25 during the early stages of biofilm development. Later, however, SBW25 formed significantly less biofilm than SBW25ΔviscA. The indication that viscosin is involved in biofilm dispersal was confirmed by chemical complementation of the mutant biofilm. Further, a fluorescent bioreporter showed that viscA expression was induced in biofilms 4 hours prior to dispersal. Subsequent detailed studies of biofilms formed in flow-cells for up to 5 days revealed that SBW25 and SBW25ΔviscA developed comparable biofilms dominated by well-defined mushroom-shaped structures. Carbon-starvation was required to obtain biofilm dispersal in this system. Dispersal of SBW25 biofilms was significantly larger than of SBW25ΔviscA biofilms after 3 hours, and importantly, carbon-starvation strongly induced viscA expression, in particular for cells that were apparently leaving the biofilm. Hence the current study points towards a role for viscosin-facilitated motility in dispersal of SBW25 biofilms.