The efficient treatment and energy recovery from salvaged microalgae have been imperative considering both environmental and economic concerns. Herein, this study proposed a biotechnological platform for converting microalgae into medium-chain fatty acids (MCFAs) through anaerobic fermentation as well as exploring the roles of different electron donors (EDs). After exploring various ED combinations, the results suggested that a single ED, especially sole ethanol stimulation, exhibited more pronounced stimulation effects on MCFA production compared to those of the hybrid ED stimulations. Furthermore, ethanol and lactic acid served as the basis for the formation of longer-chain alcohols and odd-chain fatty acids in the liquid fermentation products, respectively. The dynamics and thermodynamics analyses confirmed the distinctions in the accumulation trends and saturated compositions of main products under different ED compositions. Moreover, mechanistic investigations revealed that differences in chain elongation efficiency of ethanol and lactic acid and in the genome-based metabolic potential for the microbial systems contributed to the intricate feedback regulations of biochemical reactions in different microalgae fermentation scenarios. Overall, this study provided valuable insights for sustainable energy conversion of microalgae by orienting toward the recovery of high-value biochemicals, serving as a theoretical basis for the selection and optimization of ED before industrial application.