Historically, commercial fishers harvested Lake Superior lake herring Coregonus artedi for their flesh, but recently operators have targeted lake herring for roe. Because no surveys have estimated spawning female abundance, direct estimates of fishing mortality are lacking. The primary objective of this study was to determine the feasibility of using acoustic techniques in combination with midwater trawling to estimate spawning female lake herring densities in a Lake Superior statistical grid (i.e., a 10′ latitude × 10′ longitude area over which annual commercial harvest statistics are compiled). Midwater trawling showed that mature female lake herring were largely pelagic during the night in late November, accounting for 94.5% of all fish caught exceeding 250 mm total length. When calculating acoustic estimates of mature female lake herring, we excluded backscattering from smaller pelagic fishes like immature lake herring and rainbow smelt Osmerus mordax by applying an empirically derived threshold of −35.6 dB. We estimated the average density of mature females in statistical grid 1409 at 13.3 fish/ha and the total number of spawning females at 227,600 (95% confidence interval = 172,500–282,700). Using information on mature female densities, size structure, and fecundity, we estimate that females deposited 3.027 billion (10⁹) eggs in grid 1409 (95% confidence interval = 2.356–3.778 billion). The relative estimation error of the mature female density estimate derived using a geostatistical model—based approach was low (12.3%), suggesting that the employed method was robust. Fishing mortality rates of all mature females and their eggs were estimated at 2.3% and 3.8%, respectively. The techniques described for enumerating spawning female lake herring could be used to develop a more accurate stock–recruitment model for Lake Superior lake herring.