Producing　high-value　medium-chain　fatty　acids　(MCFAs)　via　anaerobic　fermentation　is　an　emerging　green　biotechnology　to　recover　bioenergy　from　waste-activated　sludge　(WAS).　Electron　donor　(ED)　is　a　key　driver　affecting　the　MCFA　production　profiles.　This　study　investigated　and　compared　the　potential　of　using　ethanol,　lactate,　and　a　combination　of　both　as　ED　supplies　(Co-EDs)　for　enhancing　MCFA　production　during　anaerobic　WAS　fermentation.　Adopting　ethanol　as　the　sole　ED　was　the　optimal　strategy　to　attain　the　highest　MCFA　production　(3352.7　±　564.7　mg　of　COD/L)　and　selectivity　(20.7%)　from　WAS.　Although　lactate　dosage　promoted　WAS　degradation,　more　organics　were　converted　to　short-chain　fatty　acids　(SCFAs)　rather　than　MCFAs,　resulting　in　the　lowest　MCFA　production　(1034.8　±　303.6　mg　of　COD/L)　afterward.　Lactate-　and　Co-EDs-added　systems　enriched　hydrolytic　bacteria　and　SCFA　producers　to　a　higher　level　than　the　reactor　with　an　ethanol　supplement.　Nevertheless,　the　richest　abundance　of　Clostridium_kluyveri,　a　key　MCFA　producer,　was　attained　in　the　ethanol-added　system.　Further　metatranscriptomic　analysis　mapped　all　expressed　metabolic　pathways　involved　in　MCFA　production　and　revealed　that　no　synergistic　effect　was　observed　in　the　Co-ED　system.　The　insufficient　electron　acceptor　(EA)　synthesis　at　the　initial　stage　induced　the　inefficiency　in　ED　usage　for　CE,　rendering　a　huge　waste　of　ED　(i.e.,　lactate)　via　the　competitive　acrylate　pathway.　In　comparison,　the　gene　abundance　related　to　the　CE　cycle　was　the　highest　in　the　ethanol　system.　This　study　provided　useful　information　and　insights　for　enhancing　the　MCFA　yield　by　optimizing　ED　composition　from　various　substrates.　©　2023　American　Chemical　Society.