Food-borne pathogen contamination of fresh produce represents a crucial problem in terms of food safety and economic losses. To avoid outbreaks and release of contaminated products in the market, food producers must assure that safety and control measures are followed throughout the production chain. Since traditional methods are complex and time consuming, the use of rapid and reliable methods is needed for a reproducible detection of low pathogen levels prior to packaging. To respond to this need, an indirect ELISA assay was developed to detect the presence of Listeria monocytogenes and Escherichia coli O157. Bacteria isolation procedure, antibody working concentration and limit of detection were studied and optimized to verify the presence of the two bacteria on cucumber. Incubation times for antigen (overnight, 4 °C), antibodies (60 minutes, 25 °C) and for substrate reaction (30 min, 25 °C) were selected. Results show that the ELISA method was highly sensitive with a detection limit lower than 103 CFU g−1 and relatively fast because bacteria isolation was achieved from 1 to 7 hours.