The biogeochemistry of Lake Vida brine, Antarctica, was investigated by studying the organic matter composition in a cold, isolated, subzero, hypersaline, anoxic, and aphotic brine environment. The brine of Lake Vida, encapsulated within the thick (27+ m) of Lake Vida ice, harbors an exclusively bacterial community that is metabolically active at maintenance level. Lake Vida brine is a unique analog for icy planetary worlds. Studying the organic biosignatures that are produced, processed, and transformed in Lake Vida brine can enhance our understanding of life in subzero habitats and inform our search for extant biosignatures of life on other icy worlds, such as Mars, Europa, or Enceladus. First, I investigated the standing crop of organic biosignatures in Lake Vida brine using environmental metabolomics. Analysis of the dissolved metabolites of Lake Vida brine revealed that legacy paleometabolites from past environmental conditions remained in the brine after millennia of isolation. Second, I characterized novel organic sulfones in a fraction of the dissolved organic matter in Lake Vida brine in order to further understand the ancient and modern processes that dominate the Lake Vida brine environment. Third, I investigated the particulate organic matter of Lake Vida brine using combined lipidomics and targeted metagenomics approaches in order to distinguish modern from ancient metabolites by interrogating the genetic capacity of the modern Lake Vida brine microbial community. Eukaryotic cellular detritus and legacy lipids that were indicative of a different, possibly photosynthetic, community were detected. Results indicate that Lake Vida brine contains abundant legacy organic matter in both the dissolved and particulate fraction. However, metagenomics analysis suggests that some of the lipids observed in the particulate organic matter fraction could potentially be modern. Ultimately, the presence of legacy biosignatures in the Lake Vida brine organic matter suggests that organic biosignatures of previous ecosystems may be preserved in cold, isolated ecosystems for long timescales. The prevalence of legacy in Lake Vida brine is due to the slow-growing, cold-limited lifestyle of the modern microbial assemblage. I posit that the abundance of legacy in other cold-limited, slow-growing ecosystems, on Earth and on other icy worlds, is not likely trivial and may affect the standing crop of the organic biosignatures of an environment at a given time. However, combined with metagenomics analysis, potentially achieved by space-capable sequencing technology, may allow for an integrated investigation of organic biosignatures, providing an avenue for detecting modern biosignatures, and thus, the presence of extant life