The increase of crop production along with stricter requirements on food security have augmented the demand of new and eco-friendly bactericides. Most of the bactericides used at the moment consist of persistent organic substances, representing a risk for environmental and human health. For instance, agriculture bactericides used for crop protection includes copper-based, dithiocarbamate and amide bactericides, which are not biodegradable, resulting in the necessity of further research about the production of new active principles that attack microorganisms without producing any harmful effect on human health or environment. The biosurfactant extract evaluated in this work as a bactericide, is obtained from corn steep water, a residual stream of corn wet milling industry, which is fermented spontaneously by probiotic lactic acid bacteria that possess the capacity to produce biosurfactants. In previous works, it has been demonstrated that this biosurfactant extract is able to promote the growth of Lactobacillus casei in drinkable yogurts, though its antimicrobial activity against pathogenic strains has not been evaluated at the moment. The results obtained in this work have proved that this biosurfactant extract is effective as bactericide against Pseudomonas aeruginosa and Escherichia coli, at concentrations of 1 mg/mL, opening the door to its use in agrifood formulations for reducing the use of chemical pesticides and preservatives.