Bat guano is an emergent paleoenvironmental archive and isotopic compositions of bat guano have been tied to climate, vegetation, and trophic processing in isolated studies. However, a comprehensive, multi-isotope assessment of bat guano has not been established, hindering the proliferation of guano-based paleoenvironmental research. In this study, we comprehensively examine isotopic compositions of a 12,000-year long bat guano core from Cave Springs Cave, Alabama as well as relevant isotopic data from other sources to improve our understanding of each isotope system (δ13C, δ15N, and δ2H), the source-to-sink components of guano isotope pathways, and the fractionation occurring along each multi-step isotopic pathway from sources (i.e., soil, atmosphere, rainfall) to guano formation. We find the δ13C values of guano from Cave Springs Cave primarily reflect local vegetation with a large shift that coincides with the onset of the Anthropocene (after 1950 C.E.). We also determine that the δ15N values of Cave Springs Cave guano were not associated with precipitation or any other climatic variable but instead reflect a change in bat diet and agricultural intensification during the past 600 years. Overall, these data adds to the existing body of bat guano research and supports bat guano as a reliable archive for paleoenvironmental reconstructions.