The aim of the present study was to optimise the production of a biosurfactant by a new strain of Pseudomonas cepacia CCT6659 with aid of a combination of central composite rotatable design (CCRD) and response surface methodology (RSM). The factors selected for optimisation of the growth conditions were canola waste frying oil, corn steep liquor and NaNO3 substrate concentrations. Surface tension was chosen as the response variable. All factors studied were important within the ranges investigated. The empirical forecast model developed through RSM regarding effective nutritional factors was adequate for explaining 89 % of the variation observed in biosurfactant production. Maximal reduction in surface tension of 26 mN m–1 was obtained under the optimal conditions of 2 % waste frying oil, 3 % corn steep liquor and 0.2 % NaNO3. The accumulation of isolated biosurfactant increased from 2 g L–1 to 8.0 g L–1 under these conditions, demonstrating that the factorial design is adequate for identifying the optimal conditions for biosurfactant production.