Biobeds are an effective system for minimising the risk of point-source contamination by pesticides. The original biobed system comprises a clay layer at the bottom, a biomixture layer and a grass layer on top. The composition of the biomixture, which in the original Swedish model consisted of soil: peat: wheat straw (1:1:2 by volume), determines the efficiency of the system. Since the use of peat is not environmentally or economically feasible in China, this study tested the potential of replacing it with a locally available material, spent mushroom substrate (SMS). Three biomixtures containing different SMS (Pleurotus eryngii, Flammulina velutipes and Lentinus edodes) were compared with a biomixture containing peat, as in the original Swedish design, and a control containing soil alone. The fungicide chlorothalonil and the insecticide imidacloprid were used as model pesticides in the tests. Microbial activity (measured as respiration and phenoloxidase and hydrolytic activity) and pesticide dissipation were studied. Microbial activity was higher in the three biomixtures containing SMS than in the original-type biomixture. Among the SMS biomixtures, that containing SMS from L.edodes was the most biologically active. However, pesticide dissipation was comparable in all four biomixtures and significant differences were only found between biomixtures and the soil-alone control. Based on the physicochemical characteristics, biological activity and preliminary results on pesticide degradation, SMS are suitable and can therefore be used as a substitutes for peat.