The Late Ordovician mass extinction was linked to climate cooling and glaciation of Gondwana during the terminal Ordovician Hirnantian Age (444.7–443.4 Ma). Extinction patterns have been well described for many marine taxa, but much less is known about marine microbial communities through this interval. To elucidate the structure of microbial communities in tropical marine basins through the Late Ordovician, we analyzed lipid biomarkers in thermally well preserved strata from the Taconic foreland (Anticosti Island, Canada), the Cincinnati Arch (midwestern United States), and the western continental margin (Vinini Formation, Nevada, United States). Despite clear oceanographic differences, lipid biomarker profiles show similarities between these three localities. Major shifts in biomarker distributions of Anticosti Island and the Vinini Formation, mainly hopane/sterane ratios, record changes in the balance of bacterial versus algal primary production. Bacterial contributions to sedimentary organic matter were highest during warm intervals, both before and after Hirnantian cooling. In particular, 3β-methylhopanes, likely sourced from aerobic methanotrophic bacteria, occur in high relative abundance (many times the Phanerozoic average) across Laurentia throughout most of the interval studied. 3β-methylhopane abundances also reveal an overall positive relationship with paleotemperature proxies, implying increased methane cycling during warm intervals. These results suggest that enhanced methane cycling could have provided an important positive feedback on climate during extended intervals of early Paleozoic time.