Acid mine drainage (AMD), characterized by a high concentration of heavy metals, poses a threat to the ecosystem and human health. Bioelectrochemical system (BES) is a promising technology for the simultaneous treatment of organic wastewater and recovery of metal ions from AMD. Different kinds of organic wastewater usually contain different predominant organic chemicals. However, the effect of different energy substrates on AMD treatment and microbial communities of BES remains largely unknown. Here, results showed that different energy substrates (such as glucose, acetate, ethanol, or lactate) affected the startup, maximum voltage output, power density, coulombic efficiency, and microbial communities of the microbial fuel cell (MFC). Compared with the maximum voltage output (55 mV) obtained by glucose-fed-MFC, much higher maximum voltage output (187 to 212 mV) was achieved by MFCs fed individually with other energy substrates. Acetate-fed-MFC showed the highest power density (195.07 mW/m²), followed by lactate (98.63 mW/m²), ethanol (52.02 mW/m²), and glucose (3.23 mW/m²). Microbial community analysis indicated that the microbial communities of anodic electroactive biofilms changed with different energy substrates. The unclassified_f_Enterobacteriaceae (87.48%) was predominant in glucose-fed-MFC, while Geobacter species only accounted for 0.63%. The genera of Methanobrevibacter (23.70%), Burkholderia-Paraburkholderia (23.47%), and Geobacter (11.90%) were the major genera enriched in the ethanol-fed-MFC. Geobacter was most predominant in MFC enriched by lactate (45.28%) or acetate (49.72%). Results showed that the abundance of exoelectrogens Geobacter species correlated to electricity-generation capacities of electroactive biofilms. Electroactive biofilms enriched with acetate, lactate, or ethanol effectively recovered all Cu²⁺ ion (349 mg/L) of simulated AMD in a cathodic chamber within 53 h by reduction as Cu⁰ on the cathode. However, only 34.65% of the total Cu²⁺ ion was removed in glucose-fed-MFC by precipitation with anions and cations rather than Cu⁰ on the cathode.