Pseudomonas fluorescens is a well-known biofilm former on food contact surfaces and can cause severe cross-contamination in food processing premises. This study aimed to determine the inactivation effect of low-energy X-ray on P. fluorescens planktonic cells in phosphate-buffered saline solution (PBS) and P. fluorescens biofilm cells on food-contact-surface (stainless steel). The results demonstrated that low-energy X-ray irradiation at 125 Gy inactivated 4.60 log CFU/mL and 4.21 log CFU/cm2 for P. fluorescens planktonic and biofilm cells, respectively. Based on Weibull model, low-energy X-ray achieved tR1 values of 14.8 Gy and 11.6 Gy for P. fluorescens planktonic and biofilm cells, respectively. Apart from cell inactivation, the irradiation also led to the destruction of extracellular polymeric substances (EPS) structure. Low-energy X-ray irradiation markedly damaged bacterial glucose uptake system and resulted in part loss of bacterial membrane potential and integrity. These results suggested the potential of the low-energy X-ray for inactivating P. fluorescens biofilm cells and removing EPS in food industry.