Foodborne pathogens such as Escherichia coli O157:H7, Listeria monocytogenes, and Salmonella can easily transfer from food to food contact surfaces. Methods for rapid detection of these pathogenic bacteria are important in order to avoid contamination of food. Here, we describe the detection and quantification of bacterial pathogens on contaminated surfaces by real-time PCR (RT-PCR) and culture methods using a single enrichment medium. Surfaces of wood, polypropylene, and stainless steel were inoculated with a mixed culture of E. coli O157:H7, L. monocytogenes, and S. enterica. Surfaces were sampled after an initial contact time of 10 min and after 16 h. Results indicated that after a short contact time, RT-PCR gave results similar to standard microbiological counts for each pathogen tested. However, after being left for 16 h on surfaces, the detection of these pathogens at low inoculation levels was always possible with RT-PCR, while microbiological methods failed to detect them in many cases. In conclusion, RT-PCR is more sensitive and rapid than are standard microbiological methods for the detection of bacterial pathogens on food contact surfaces.