This study developed a microbial validation method for radio frequency (RF) pasteurization of low-moisture food powders. Wheat flour with water activity of 0.45 ± 0.02 was used as a model. In this study, heat resistance parameters (D- and z-values) of Salmonella Enteritidis PT 30 (S. Enteritidis) and its potential surrogate Enterococcus faecium NRRL B-2354 (E. faecium) in wheat flour were determined. The results showed that, while both microorganisms yielded the similar z-values, E. faecium was more heat-resistant than S. Enteritidis. For process validation, a 5-g pack of wheat flour inoculated with either microorganism was placed in the geometric center of 3 kg wheat flour and subjected to various processing times of up to 39 min in a 27 MHz RF unit. The inactivation kinetics matched but yielded slightly greater reduction than pasteurization modeled from measured temperature profiles and microbial thermal resistance parameters. This investigation concluded that E. faecium is a valid surrogate for Salmonella in wheat flour. A conservative validation can be obtained by inoculated pack protocol. RF heating technology has potential for pasteurizing wheat flour.