Background: Certain microorganisms during the preparation of substrate for growing Agaricus bisporus are of great interest for the conversion of organic material and for the excretion of a wide range of metabolites with growth regulation activities that may affect the mycelial growth processes and mushroom yield. Adding beneficial microorganisms in the substrate may be a biotechnological alternative to optimize A. bisporus cultivation. Objective: Isolate and evaluate thermophilic microorganisms from self-heating pasteurized substrate with growth-promoting effects on A. bisporus cultivation. Methods: Different microorganisms were isolated and selected at 45 and 55 °C. They were tested for siderophore production, 1-octen-3-ol consumption, and phosphate solubilization in coculture with A. bisporus to determine their growth effects on potato dextrose agar (PDA) and on sterile pangola grass (Digitaria eriantha). Results: Of the 106 microorganisms isolated, 88 % were able to grow in the presence of 1-octen-3-ol, while 1 % had the capacity to produce siderophores, and 55 % had the ability to solubilize phosphate. The strains Bacillus hisashii ECS-B-65, B. licheniformis ECS-B-78, Rhizomucor pusillus ECS-710 and ECS-712, Aspergillus fumigatus ECS-709, and Thermomyces sp. ECS-711 were found to have a positive effect on A. bisporus mycelial growth.