The main objective of this work is to present the optimization of the biotransformation of R-(+)-limonene and (-)-beta-pinene aiming at the production of alpha-terpineol by strains of fungal and yeasts previously isolated by our research group using the methodology of experimental design. New optimized experimental data on alpha-terpineol production by the biotransformation of R-(+)-limonene and (-)-beta-pinene using newly isolated microorganisms are reported in this work. Conversion of about 1,700 mg/L was achieved when R-(+)-limonene was used as substrate and the newly isolated strain 05.01.35 as microorganism at the central point of the experimental design, corresponding to a substrate concentration of 1.75%, mass of inoculum of 2 g, and substrate to ethanol volume ratio of 1:1. The same experimental condition led to higher conversions when (-)-beta-pinene was used as substrates and the strains coded as 04.05.08 and 01.04.03 as microorganism. Here, conversions of about 770 mg/L were achieved.