Abstract: The technology of the production of medium-chain fatty acids (MCFAs) from waste activated sludge via carbon chain elongation in biological fermentation represents a novel and highly promising commercial pathway for the high-value utilization. This study focused on optimizing the initiation phase of chain elongation fermentation, by systematically investigating the effects of the initial molar ratio of electron donor (ED) to electron acceptor (EA) on the startup performance. Ethanol and sludge fermentation liquor were used as ED and EA, respectively. The results identified an optimal ethanol dosing strategy at an initial ED/EA ratio of 4∶1. Under this condition, caproic acid is the main product of MCFAs. emerged as the dominant MCFA product, rapidly accumulating to 11.5 g/L (calculated as COD, same below) within 10 days, accounting for 30.3% of the fermentation broth components. The initial accumulation of caproic acid followed a modified Gompertz kinetic model, with the highest production potential and productivity observed at the ED/EA ratio of 4∶1, reaching 11.85 g/L and 3.06 g/(L·d), respectively. Under this dosing strategy, high-throughput sequencing revealed 12 dominant bacterial genera with relative abundances exceeding 1%, including Proteiniphilum, Fermentimonas, D8A-2, and Paraclostridium. Among them, Fermentimonas and Acetomicrobium were identified as the key biomarkers, facilitating the degradation of substrates such as ethanol, proteins, and amino acids, as well as the generation of acetate-critical precursors for chain elongation metabolism. These genera acted synergistically with other community members to drive MCFA synthesis. To mitigate product inhibition associated with high initial ED/EA ratios, a subsequent feeding strategy with a reduced ED/EA ratio of 2∶1 was recommended. This study provided theoretical insights and empirical data support for achieving the high-value valorization of waste activated sludge.