As a multifunctional material, biochar is considered a potential adsorbent for removing heavy metals from wastewater. Most biochars with high adsorption capacities have been modified, but this modification is uneconomical, and modifying biochar may cause secondary pollution. Thus, it is necessary to develop an efficient biochar without modification. In this study, spent P. ostreatus substrate and spent shiitake substrate were used as the raw materials to prepare biochar. Then, the physicochemical properties of the biochars and their removal efficiencies for Pb(II) were investigated. The results showed that the physicochemical properties (e.g., large BET surface area, small pore structure and abundant functional groups) contributed to the large adsorption capacity for Pb(II); the maximum adsorption capacities were 326 mg g-1 (spent P. ostreatus substrate-derived biochar) and 398 mg g-1 (spent shiitake substrate-derived biochar), which are 1.6-10 times larger than those of other modified biochars. The Pb(II) adsorption data could be well described by the pseudo-second-order kinetic model and the Langmuir model. This study provides a new method to comprehensively utilize spent mushroom substrates for the sustainable development of the edible mushroom industry.