The effect of two food intrinsic factors (fat content, matrix structure) on the radio frequency (RF) inactivation of Salmonella Typhimurium and Listeria monocytogenes was investigated in dairy model systems: i.e., liquids with 0.08% and 5.9% fat and gels with 0.08% and 5.9% fat. In the liquid, higher fat content resulted in higher inactivation for both microorganisms. In gels, inactivation rate was higher with the lower fat content, while opposite behaviour occurred for L. monocytogenes above 70 °C. A gel matrix resulted in higher inactivation of S. Typhimurium, while complex temperature-dependent behaviour was present for L. monocytogenes. Sublethal injury was most significant for L. monocytogenes at 5.9% fat content and high temperatures. Overall, fat content and matrix structure impacted the microbial inactivation during RF heating and should be taken into account when designing novel RF heating food decontamination strategies. Industrial relevanceRadio frequency (RF) heating as a food processing technology has seen increased industrial application in recent years. The technology allows a more homogeneous heating of food products and also enables the treatment of low-moisture foods. The increased knowledge on the influence of fat content and food structure on RF thermal inactivation will lead to the development of improved and more efficient treatments.