Natural history collections (NHCs) are an underutilized resource to investigate anthropogenic influences and assess temporal trends in mammal gut microbiome diversity. Quantifying microbiome diversity can provide critical insights into species health, ecology, and evolution. This study leverages these collections to explore the impacts of anthropogenic, dietary, and climatic factors on the gut microbiome diversity of fluid-preserved rodent and bat specimens. Utilizing advanced genomic techniques, we extracted fragmented DNA from formalin-fixed specimens housed at the University of Massachusetts Amherst Mammalogy collections. Our analysis aimed to discern historical differences in rodent and bat gut microbiomes and examine the effects of anthropogenic activities, dietary variations, and climatic conditions on these microbiomes. Our findings confirmed that human-impact and diet significantly influence microbial diversity in gut microbiomes. Samples from more urbanized areas displayed lower microbial diversity. Surprisingly, we found a low abundance of Proteobacteria in the bat specimens in comparison to contemporary studies. Given the extensive geographic distribution and zoonotic relevance of rodents and bats in human pathogen transmission, understanding their microbial responses to environmental changes is crucial. Our results underscore the potential of museum collections to provide insights into the evolutionary and ecological dynamics of host-microbiome interactions in the face of anthropogenic and climatic shifts.