This study explored the differences in the microbial diversity and physicochemical properties of mushroom residue and cow manure to provide a theoretical basis for the use of mushroom residue as cow bedding. High-throughput sequencing was used to analyze the bacterial community composition of mushroom residue and cow manure bedding and determine the physical and chemical properties of these different bedding materials. The results showed that the bacterial communities in the two types of bedding materials could be categorized into 6 classes, 13 orders, 32 families, and 48 genera. The dominant genus in the mushroom residue bedding samples after use by cows was Lactobacillus (36.37%) followed by Corynebacterium (22.15%). The dominant group in the cow manure bedding samples after use was "other" (28.8%), followed by Solibacillus (8.76%). The different bedding materials contained varying number of bacterial species. After use, 499 bacterial species were present in the cow manure bedding, while only 345 bacterial species were present in the mushroom residue bedding. The utilization rate of the mushroom residue bedding by dairy cows was 79%, whereas that of the cow manure bedding was 61%. The results of this study provide a theoretical basis for the application of mushroom residue bedding for dairy cows.