Foodborne pathogens are of significant concern in the agrifood industry and the development of associated rapid detection and identification methods are of major importance. This paper describes the novel use of resolution-optimized prism-based surface plasmon resonance imaging (RO-SPRI) and data processing for the detection of the foodborne pathogens Listeria monocytogenes and Listeria innocua. With an imaging spatial resolution on the order of individual bacteria (2.7 AE 0.5 mm  7.9 AE 0.6 mm) over a field of view 1.5 mm 2 , the RO-SPRI system enabled accurate counting of individual bacteria on the sensor surface. Using this system, we demonstrate the detection of two species of Listeria at an initial concentration of 2  10 2 CFU mL À1 in less than 7 hours. The surface density of bacteria at the point of positive detection was 15 AE 4 bacteria per mm 2. Our approach offers great potential for the development of fast specific detection systems based on affinity monitoring.