Lipid biomarkers are recalcitrant organic natural products that have potential to provide information about the major contributors to sedimentary organic matter in past environments, about geochemical conditions during deposition, and about the thermal history of rocks. Because tiny, non-mineralizing cells of microbes and of Paleozoic primary producers have low fossilization potential, lipid biomarkers are one of the few ways to acquire information about the base of marine food webs in deep time. Further, the intricate linkage between microbial communities and environmental conditions (nutrients, dissolved oxygen), provides greater context for interpreting the macrofossil record. The Late Ordovician has received the most attention as a mass extinction linked to climate change. We compiled stratigraphic lipid biomarker records of microbial communities to better understand both the baseline and response to changing environmental conditions in the Late Ordovician. This information will be of use in assessing the causes of climatic change, extinction, and impacts of inferred cooling on marine geochemistry. We present results from thermally well-preserved strata from the Laurentian Taconic foreland (Anticosti Island), mid-continent (Cincinnati Arch, eastern Iowa), and western continental margin (Vinini Formation), as well as the Baltic shelf (Estonia, Sweden). Lipid biomarker distributions, primarily hopane/sterane ratios, document strong relationships between nutrient availability and the balance of primary production between bacteria and algae, with bacteria favored in oligotrophic waters and algae predominating in waters influenced by upwelling or runoff. The Hirnantian glacial maximum presents both spikes in hopane/sterane and a decrease in average ratio below the pre-Hirnantian mean, perhaps related to disruption of eukaryotic productivity and increased nutrient availability, respectively. Compounds derived from aerobic methanotrophic bacteria (3beta-methylhopanes) occur in high relative abundance across the paleotropics throughout the studied interval. The positive relationship between aerobic methanotroph markers and paleotemperature proxies implies increased methane cycling during warm intervals, an important positive feedback on climate during extended intervals of Early Paleozoic time