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Different bioproducts can be obtained by changing operative condition of biotechnological process, and this bioprocess aspect is a significant approach to be adopted on industrial scale leading to the creation of new natural aroma. Thus, this study aimed to investigate the culture conditions and optimization of the biotransformation of limonene into limonene-1,2-diol using Pestalotiopsis mangiferae LaBMicrA-505 obtained from the Brazilian Amazon. The study started with the investigation of the establishment of culture, followed by optimization of the conditions for biotransformation of R-(+)-limonene to limonene-1,2-diol, using shake flasks. The fresh biomass of P. mangiferae LaBMicrA-505 obtained in liquid media supplemented with yeast-malt extract under with 72 h (stationary phase) performed better diol productivity when compared to other biomasses. Finally, in the modeling of contour plots and surface responses of a central composite design, the use of 4 g l - 1 biomass, 2% of the substrate at 24 °C, 120 rpm, and pH of 6.0 could maximize the production of limonene-1,2-diol, accumulated up to 98.34 ± 1.53% after 96 h of reaction. This study contributed to identified operational condition for the R-(+)-limonene bioconversion scale-up. The endophytic fungus P. mangiferae LaBMicrA-505 proved to be a potent biocatalyst to biotechnologically produce limonene-1,2-diol, an aroma compounds with interesting bioactive features that up to now has been manufactured by extraction from plants with long and not environmentally friendly procedures., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)