The inactivation of biofilms formed by pathogenic bacteria on ready-to-eat and minimally processed fruits and vegetables by nonthermal processing methods is critical to ensure food safety. Pulsed ultraviolet (PUV) light has shown promise in the surface decontamination of liquid, powdered, and solid foods. In this study, the antimicrobial efficacy of PUV light treatment on nascent biofilms formed by Escherichia coli O157:H7 and Listeria monocytogenes on the surfaces of food packaging materials, such as low-density polyethylene (LDPE), and fresh produce, such as lettuce (Lactuca sativa) leaves, was investigated. The formation of biofilms on Romaine lettuce leaves and LDPE films was confirmed by crystal violet and Alcian blue staining methods. Inactivation of cells in the biofilm was determined by standard plating procedures, and by a luminescence-based bacterial cell viability assay. Upon PUV treatment of 10 s at two different light source to sample distances (4.5 and 8.8 cm), viable cell counts of L. monocytogenes and E. coli O157:H7 in biofilms on the lettuce surface were reduced by 0.6-2.2 log CFU mL(-1) and 1.1-3.8 log CFU mL(-1), respectively. On the LDPE surface, the efficiency of inactivation of biofilm-encased cells was slightly higher. The maximum values for microbial reduction on LDPE were 2.7 log CFU mL(-1) and 3.9 log CFU mL(-1) for L. monocytogenes and E. coli O157:H7, respectively. Increasing the duration of PUV light exposure resulted in a significant (P < 0.05) reduction in biofilm formation by both organisms. The results also revealed that PUV treatment was more effective at reducing E. coli biofilms compared with Listeria biofilms. A moderate increase in temperature (~7-15°C) was observed for both test materials. PUV is an effective nonthermal intervention method for surface decontamination of E. coli O157:H7 and L. monocytogenes on fresh produce and packaging materials.