Biopurification systems designed for pesticide treatment are a source of diverse bacteria with environmental and biotechnological applications, including Pseudomonas marginalis C9, which has been reported as a biosurfactant-producing bacterium. The optimization of biosurfactant produced from P. marginalis C9 to enhance the solubility of a hydrophobic pesticide of environmental interest was investigated. The response surface methodology (RSM) was used to optimize the combined effect of the initial pH (5–9), agitation (100–300 rpm), and temperature (24–32 °C) on biosurfactant production. A DASbox® automated mini-bioreactor system was used to evaluate the critical factors in biosurfactant production using a full factorial design (FFD). The results showed that the optimal culture conditions using RSM were a pH of 8.5, a temperature of 25 °C, and agitation at 200 rpm. The extraction yield of the biosurfactant was 7.40 g L−1, the surface tension was reduced to 27.45 mN m−1, and the critical micelle concentration (CMC) was 48.9 mg L−1. The FFD analysis indicated that a high agitation rate (300 rpm) strongly influenced the biosurfactant activity, regardless of the inlet oxygen supply (0.5–1.5 vvm). The rhamnolipid increased the water solubility of chlorpyrifos by 11.2- and 21.7-fold at the CMC and twice the CMC, respectively.