Fusel oil is a mixture of higher alcohols that are formed during fermentation, and the main constituents are isoamyl alcohol and isobutanol. Although their presence in fermented musts is detrimental to the distillation process and ethanol quality, the aforementioned higher alcohols are widely used, especially in the fine chemical industry. On the other hand, the quality and quantity of fusel oil depend on various factors, including raw materials and fermentation conditions. The aim of this study was to investigate the effects of pH, refrigeration, and supplementation on the formation of isoamyl alcohol and isobutanol during the fermentation of molasses must in a microdistillery. The fermentations were conducted in batches that were fed with 25 °Brix must and 25% v/v commercial dry yeast for 10 hours. A complete 2³ factorial design was used to assess the effects of the studied factors and their interactions on the response variables: fermentation efficiency (nf), process efficiency (np), ethanol productivity (P), substrate-to-cell conversion factor (YX/S), isoamyl alcohol produced (A), isobutanol produced (B) and the A/B Ratio between these alcohols. For statistical analysis, analysis of variance (ANOVA) and Tukey’s test were used for mean comparisons. The results of the substrate-to-cell conversion factor (YX/S) indicated good yeast performance under different fermentation conditions. The interaction effects among the evaluated factors significantly influenced the formation of isoamyl alcohol and isobutanol, as well as the A/B Ratio.