Biosurfactant produced from Pseudomonas aeruginosa DSVP20 was evaluated for its potential to disrupt Candida albicans biofilm formed on polystyrene (PS) surfaces in this investigation. P. aeruginosa DSVP20 exhibited optimum production of biosurfactant (5.8 g L-1) after 96 h of growth with an ability to reduce surface tension of the aqueous solution from 72 to 28 mN m(-1). Analysis of purified biosurfactant with FT-IR, H-1 and C-13 NMR and MALDI-TOF MS revealed it to be di-rhamnolipid (RL-2) in nature. Biofilm disrupting ability of RL-2 (0.16 mg mL(-1)) on Candida cells when checked using XTT reduction assay revealed that about 50 % of the cells remain adhered to 96-well plate after 2 h of treatment, while up to 90 % reduction in pre-formed C. albicans biofilm on PS surface was observed with RL-2 (5.0 mg mL(-1)) in a dose-dependent manner. Microscopic analyses (SEM and CLSM) further confirm the influence of RL-2 on disruption of Candida biofilm extracellular matrix on PS surface which can be exploited as a potential alternative to the available conventional therapies.