This work assesses the in vitro antimicrobial activity of an aqueous olive mill waste extract (AE-2) on the growth of diverse cocktails of foodborne pathogens species (Listeria monocytogenes, Staphylococcus aureus, Escherichia coli, and Salmonella Enterica). The effects were evaluated by Response Surface Methodology, using a two-block (D-optimal and full factorial) sequential design, with two independent variables (hydroxytyrosol concentration 0–3000 ppm and pH 3.5–6.5) and the percentage of inhibition (%I) as the dependent variable. S. Enterica and E. coli behaviours were similar but different from L. monocytogenes and S. aureus. The models predicted the complete inhibition of the four foodborne pathogen cocktails in the region defined by 3.80–3.87 pH and 1200–1314 ppm hydroxytyrosol. Within the experimental region, the model showed the best predictions for L. monocytogenes and the worst for S. Enterica, but the errors never exceeded 46%. This study could promote the use of olive by-products as natural preservatives in the food industry, especially in acidic matrices. •Response Surface Methodology was effective to evaluate the antimicrobial effect of olive mill waste extract.•S. Enterica and E. coli behaviours were similar but different from L. monocytogenes and S. aureus.•The models predicted the complete inhibition of the four foodborne pathogen cocktails.•The model showed the best predictions for L. monocytogenes and the worst for S. Enterica.