Human pathogens can develop biofilm structures on different artificial substrates common in the food industry. In this study, we investigated the inactivation efficacy of low-energy X-ray irradiation on Escherichia coli O157:H7, Salmonella Typhimurium, and Listeria monocytogenes biofilms on food contact surfaces, including polyvinyl chloride (PVC), stainless steel with finish 2B (STS 2B), and Teflon. The numbers of viable cells in biofilms on all test coupons were significantly (p < 0.05) reduced as the X-ray dose increased. Interestingly, different biofilm inactivation levels were observed relative to various material surfaces. Teflon showed the lowest D5d (dose required for a 5-log reduction in cell count) values among three groups of coupons, whereas PVC exhibited higher D5d values than the other two coupons. The mechanism of the X-ray antibiofilm effect was identified through the measurement of extracellular polymeric substances (EPS) in biofilms. X-ray irradiation could remove exopolysaccharides, which are major component of EPS and the removal rate increased with increasing irradiation dose. The analyses also confirmed that the disintegration of EPS was strongly related to the trends of biofilm inactivation on different coupon surfaces. This study is the first to demonstrate that X-ray irradiation effectively inactivates major foodborne pathogen biofilms on various food contact surfaces and to evaluate its antibiofilm mechanisms to enhance safety in the food processing industries.