The aim of this study was to evaluate the role of acetate (C2) on n-butyric (C4) and n-caproic acid (C6) production, using fermented sugarcane molasses as a substrate. Inoculum sludge was acclimatized in a continuous upflow anaerobic sludge blanket reactor, where ethanol (EtOH) and acetate (C2) were administered at a ratio of 6:1 (g COD:g COD). Organic load rate increased gradually from 2.0 g COD L−1d-1 to 3.3, and 5.0 g COD L−1d−1, for 157 days. The acclimatized sludge was used as the inoculum in six batch assays fed with fermented molasses (ethanol-rich source) and C2 at EtOH:C2 ratios, g COD:g COD of 2:1; 4:1; 6:1, 8:1, and 10:1. A control assay was performed without C2 (WA). The EtOH:C2 ratio was more favorable toward C6 production with a ratio of 10:1, in which a C6 yield was observed as well as accumulated production of 0.4 g COD g COD−1 and 7.9 g COD L−1, respectively. Conversely, the EtOH:C2 ratio of 2:1, and the WA, resulted in C6 concentrations of 1.4 g COD L−1 and 4.3 g COD L−1, respectively. These findings confirmed that substrate composition control is essential for driving fermentation processes toward high medium-chain carboxylic acid conversion rate.