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<i>Hypsizygus marmoreus</i> has abundant proteins and is a potential source for the development of bioactive peptides. However, currently, the research on the bioactive components of <i>H. marmoreus</i> mainly focuses on polysaccharides, and there is no relevant research on the preparation of bioactive peptides. In this article, an ultrasound-assisted extraction method was used to extract proteins from <i>H. marmoreus</i>, and then, four peptides with different molecular weight ranges were prepared through protease hydrolysis and molecular classification. The antioxidant and antibacterial activities were also studied. Under the optimal conditions, the extraction rate of <i>H. marmoreus</i> proteins was 53.6%. Trypsin exhibited the highest hydrolysis rate of <i>H. marmoreus</i> proteins. The optimal parameters for enzymatic hydrolysis were a substrate concentration of 3.7%, enzyme addition of 5700 U/g, pH value of 7, extraction temperature of 55 °C, and time of 3.3 h. Under these conditions, the peptide yield was 59.7%. The four types of <i>H. marmoreus</i> peptides were prepared by molecular weight grading. Among them, peptides with low molecular weight (<1 kDa) had stronger antioxidant and antibacterial activities. This study provides a theoretical basis for the efficient preparation of <i>H. marmoreus</i> peptides and the development of antioxidant and antibacterial peptide products.