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This research presents the increase of the Trichoderma harzianum production process in a biotechnology company. The NOBA (Near-Orthogonal Balanced arrays) method was used to fractionate a mixed-level factorial design to minimize costs and experimentation times. Our objective is to determine the significant factors to maximize the production process of this fungus. The proposed 213242 mixed-level design involved five factors, including aeration, humidity, temperature, potential hydrogen (pH), and substrate; the response variable was spore production. The results of the statistical analysis showed that the type of substrate, the air supply, and the interaction of these two factors were significant. The maximization of spore production was achieved by using the breadfruit seed substrate and aeration, while it was shown that variations in pH, humidity, and temperature have no significant impact on the production levels of the fungus.