Removal of antibiotics and bacteria from wastewater is an effective approach to test the efficacy of catalyst and thus address the issues of water contamination. In this study, a stable magnetic Mg/Mn-layered double oxide-doped biochar composite (MgMnLDO-MBC) was prepared using Lentinula edodes substrate. This composite could further effectively activate the persulfates (PS), such as peroxymonosulfate (PMS) and peroxydisulfate (PDS). MgMnLDO coupling endowed BC and magnetic biochar (MBC) with enhanced surface areas, adsorption sites, and free radicals, which facilitated the improvement of catalytic activity of MgMnLDO-MBC/PS system toward pollutants. MgMnLDO-MBC/PMS system could remove 90.8% of ciprofloxacin (CIP) with 2.8 times the removal efficiency of MBC/PMS system. Density functional theory (DFT) calculation and intermediate analysis showed that the synergistic effect of nucleophilic, electrophilic, and radical active species-mediated reactions improved the removal performance of MgMnLDO-MBC/PMS system. MgMnLDO-MBC/PDS system could effi-ciently kill 97.6% Escherichia coli and 99.7% Staphylococcus aureus, and destroy the existing biofilms. The effects of composite concentration, PMS loading, pH, temperature, common anions, and humic acid on removal efficiency were also evaluated. After five consecutive cycles, MgMnLDO-MBC/PS system still exhibited high cata-lytic performance toward CIP removal and bactericidal ability. Results of quenching and electron spin resonance (ESR) showed that center dot SO, center dot OH, 1O2, and charge transfer were together involved in CIP removal, with center dot SO playing a major role. Furthermore, the double-layered metal oxide (LDO) and MBC present in MgMnLDO-MBC composites exhibited a strong synergistic effect in the catalytic process, which resulted in higher reactivity and lower leaching rate of metal ions. In this study, an environment-friendly catalyst was prepared using industrial waste and its efficiency for removal of antibiotics and bacteria from wastewater was verified.