Technical Abstract: Bacteriophage Listex P100 (phage P100) was approved by the U.S. Food and Drug Administration and U.S. Department of Agriculture's Food Safety and Inspection Service for Listeria monocytogenes control on both raw and ready-to-eat food products. In this article, we present the proof of concept on the influence of phage dose, phage contact time, and storage temperature on the listericidal activity of phage P100 in reducing the L. monocytogenes loads on the surface of fresh channel catfish fillet. The fresh catfish fillet samples were surface inoculated with approximately 4.3 log(10) colony forming units (CFU)/g of a two serotype mix (1/2a and 4b) of L. monocytogenes cells and then surface treated with phage P100. L. monocytogenes reduction was influenced by phage contact time and phage dose regardless of higher or lower temperature regimes tested on catfish fillet. The reduction in L. monocytogenes loads (p < 0.05) with the phage P100 dose of 2 x 10(7) plaque forming units (PFU)/g (7.3 log(10) PFU/g) was 1.4-2.0 log(10) CFU/g at 4 degrees C, 1.7-2.1 log(10) CFU/g at 10 degrees C, and 1.6-2.3 log(10) CFU/g at room temperature (22 degrees C) on raw catfish fillet. The phage contact time of 30 min was adequate to yield greater than 1 log(10) CFU/g reduction in L. monocytogenes, whereas 15 min contact time with phage yielded less than 1 log(10) CFU/g reduction in L. monocytogenes loads on catfish fillet. Phage P100 titer was stable on catfish fillet samples, and overall reductions in L. monocytogenes counts were still maintained over a 10-day shelf life at 4 degrees C or 10 degrees C by phage P100 treatment. These findings illustrate the effectiveness of an alternative generally recognized as safe antimicrobial such as bacteriophage Listex P100 in quantitatively reducing L. monocytogenes from fresh catfish fillet surfaces.