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Products based on Trichoderma spp. are widely used in the control of soil diseases. The objective of this work was to determine the effect of two methods of wetting the rice (M1 and M2), two different bag packing positions in the growth room, and four different areas of microporous tape as part of the production optimization process of Trichoderma asperellum. The adopted design was factorial (2 x 2 x 4) with three repetitions.Initially, the amount of water to be used in the M2 method was adjusted, and the volumes of 0, 80, 100, 120, 140, 160 and 180 ml were tested, and, then, those were evaluated by means of two note scales, one for the development of mycelium and the other for sporulation. The variables colonized rice weight (CRW) and colony forming units (CFU) were also evaluated in two experiments, for both methods. By varying the amount of water in the bag, it is concluded that the fungus is dependent on a higher percentage of moisture in the substrate, with significant differences 1 Universidade Federal de Mato Grosso do Sul (UFMS) ─ Campus Chapadão do Sul, Rodovia MS 306, km 105, Caixa Postal 112 ─ CEP 79560000, Chapadão do Sul, MS, Brasil. 2 Núcleo de Produção de Insumo Orgânico na Propriedade para uso próprio sem fins de comercialização, Fazenda Selia, GO, Brasil. 3 Universidade Federal de Paraíba (UFPB), Campus Mamanguape, PB, Brasil. 4 Universidade Estadual de Rio Grande do Sul (UERGS), Cachoeira do Sul, RS, Brasil. 5 Autor para correspondência: j51173@yahoo.com. Acta Biológica Catarinense 2020 Jul-Set;7(3):46-58 Recebido em: 8 mar. 2019 Aceito em: 13 jul. 2020 Otimização do método de produção massal de Trichoderma asperellum (Ascomycota: Hypocreaceae) em arroz 47 Acta Biológica Catarinense. 2020 Jul-Set;7(3):46-58 (P<0.05). When analyzing the ANOVA, highly significant differences were noticed (P<0.01) for the double and triple interactions between the tested factors. The M2 method allowed a greater time saving, also providing superior values for CRW and CFU, combined with the horizontal position and with a 20 cm2 microporous tape area. This combination promoted the further development of T. asperellum fungus under the conditions tested.