The functional role of lactate (HLac), as a co-substrate along with glucose (Glu) as well as an electron donor for the synthesis of caproic acid (HCa), a medium chain fatty acid (MCFAs) was studied. A varied HLac and Glu ratios were thus investigated in fed-batch anaerobic reactors (R1–R5) operated at pH 6 with a heat-treated anaerobic consortium. R1 and R5 were noted as controls and operated with sole Glu and HLac, respectively. Strategically, ethanol (HEth) was additionally supplemented as co-electron donor after the production of short chain carboxylic acids (SCCAs) for chain elongation in all the reactors. The reactor operated with HLac and Glu in a ratio of 0.25:0.75 (1.25 g/L (HLac) and 3.75 g/L (Glu)) showed the highest HCa production of 1.86 g/L. R5 operated with solely HLac yielded propionic acid (HPr) as the major product which further led to the higher valeric acid (HVa) production of 1.1 g/L within the reactor. Butyric acid (HBu) was observed in R1, which used Glu as carbon source alone indicating the importance of HLac as electron co-donor. Clostridium observed as the most dominant genera in shotgun metagenome sequencing in R2 and R3, the reactors that produced the highest HCa in comparison to other studied reactors. The study thus provided insight into the importance of substrate and electron donor and their supplementation strategies during the production of MCFAs.