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Abstract Background Different organic and inorganic bedding materials can be used in dairy farms. Among organic materials, there is an increasing interest in alternative substrates based on recycled manure solids (RMS). Microbiological analyses are crucial to monitor the microbial load and evaluate the presence of pathogens impacting animal welfare and health. However, logistic factors may hamper the possibility of immediately sending fresh samples to the laboratory, requiring storage in cooled conditions before analysis. Methods We assessed the impact of sample refrigeration and freezing of different organic and inorganic bedding substrates including separated raw manure solids (SRMS), anaerobically digested manure solids (ADMS), and new sand (NS), on the total bacterial count (TBC) and on different microbial classes. Results The TBC was higher in fresh NS and ADMS than in refrigerated and frozen samples of the same substrates; in addition, the TBC of ADMS was higher in refrigerated than frozen samples. The TBC of SRMS did not change significantly with refrigeration and freezing. Freezing reduced the total Gram-negative bacterial count more than refrigeration in all substrates. In fresh NS, Gram-negatives were higher than in both refrigerated and frozen NS. Escherichia coli counts were significantly lower in frozen than in refrigerated SRMS. However, both refrigeration and freezing of ADMS resulted in no E. coli growth. The coliform counts were also lower in frozen than refrigerated NS and SRMS. Frozen NS and ADMS showed lower counts compared to refrigeration for Gram-negative bacteria other than E. coli and coliforms. On the other hand, cold storage did not significantly impact the streptococci and streptococcus-like organisms (SSLO) count of all evaluated bedding substrates. Conclusion Refrigeration and freezing affect the bacteriological results of bedding substrates, with freezing generally leading to lower counts than refrigeration. Whenever possible, preference should be given to analyzing fresh bedding samples, however, when necessary, refrigeration would be recommended over freezing, while acknowledging that the measured bacterial load might underestimate the actual microbial content.