Commercial production of white button mushrooms (Agaricus bisporus) requires a specialized growth substrate prepared from composted agricultural by-products. Because horse and poultry manures are widely used in substrate formulations, there is a need to determine the extent to which the composting process is capable of eliminating human pathogens. In this study, partially composted substrate was inoculated with a pathogen cocktail (log 10⁶ to 10⁸ CFU/g) containing Listeria monocytogenes, Escherichia coli O157:H7, and Salmonella. Pathogen and indicator-organism reductions were followed at temperatures that typically occurred during a standard 6-day phase II pasteurization and conditioning procedure. Controlled-temperature water bath studies at 48.8, 54.4, and 60°C demonstrated complete destruction of the three pathogens after 36.0, 8.0, and 0.5 h, respectively. Destruction of L. monocytogenes and E. coli O157:H7 at 54.4°C occurred more slowly than E. coli, total coliforms, Enterobacteriaceae, and Salmonella. Microbial reductions that occurred during a standard 6-day phase II pasteurization and conditioning treatment were studied in a small-scale mushroom production research facility. After phase II composting, E. coli, coliforms, and Enterobacteriaceae were below detectable levels, and inoculated pathogens were not detected by direct plating or by enrichment. The results of this study show that a phase II composting process can be an effective control measure for eliminating risks associated with the use of composted animal manures during mushroom production. Growers are encouraged to validate and verify their own composting processes through periodic microbial testing for pathogens and to conduct studies to assure uniform distribution of substrate temperatures during phase II.