| Summary: | White button mushroom—<i>Agaricus bisporus</i> (J.E.Lange) Imbach—is among the most popular cultivated mushrooms worldwide. The most serious challenge in industrial mushroom production is the green mold disease caused by <i>Trichoderma</i> species. Our aim was to isolate and examine bacterial strains from mushroom casing material for their potential use as biocontrol agents. Twenty-seven bacterial strains were isolated and tested against mold pathogens of white button mushroom. The <i>Bacillus velezensis</i> strain SZMC 25431 was selected for further examination and tested under simulated <i>Agaricus</i> cultivation conditions against <i>T. aggressivum</i> SZMC 23834 in a 1200-L Fitotron SGC120 standard plant growth chamber. Our results showed that the bacterial treatment was effective against the pathogen in all cases, but the best results were achieved at an application concentration of 10<sup>5</sup> cells mL<sup>−1</sup>. Industrial-scale experiments were also carried out in <i>Agaricus</i> growing houses with a bearing surface of 480 m<sup>2</sup>: the bacterial suspension was mixed in water tanks applied for daily irrigation. The results suggest that the bacterial treatment may even increase the crop yield of <i>A. bisporus</i>. Based on our results, we concluded that the selected <i>B. velezensis</i> strain may potentially be used for biological and integrated treatment in <i>Agaricus</i> cultivation.
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