Fishmeal from small marine pelagic fishes reduces their availability for marine wildlife forage and artisanal fishing catches that support food security in lower income coastal nations. Fishmeal is primarily used in feeds for aquaculture, the world’s fastest-growing food sector. Replacing fishmeal in aquafeeds with more environmentally responsible alternative ingredients can help feed aquaculture transition to more sustainable production methods. Protein from defatted marine microalga, Nannochloropsis sp., produced alongside polyunsaturated fatty acids (PUFAs) for the nutraceutical market lacks a comprehensive open-access analysis of environmental impacts of producing these products from biorefineries. This study compared life cycle impacts (global warming potential, water use, land use, marine eutrophication potential, freshwater eutrophication potential, and biotic resource use) of protein from fishmeal produced in a small pelagic fish biorefinery to protein from defatted Nannochloropsis meal. We conducted an attributional life cycle assessment using primary data provided by Cellana LLC to model biomass cultivation and harvesting at the Kona Demonstration Facility (Hawaii, USA) and literature data to model the downstream processing of biomass into a high-protein fishmeal replacement for the aquafeed market and concentrated PUFAs for the nutraceutical market. Material and energy inputs from a Nannochloropsis biorefinery included 2 harvesting scenarios (wet and dry biomass) and 2 scenarios for oil extraction and processing (i.e., oil fractionation and concentration of PUFAs): solvents or supercritical carbon dioxide. Results for aquafeed protein from defatted Nannochloropsis were that cultivation processes had the largest overall effect for all scenarios; urea and pure liquid carbon dioxide were environmental hot spots; and the processing scenario involving dry biomass followed by oil extraction and oil processing with solvent had significantly lower environmental impacts than protein from fishmeal from a small pelagic fish biorefinery for global warming potential, water use, marine eutrophication potential, freshwater eutrophication potential, and biotic resource use, but not for land use. These results suggest that aquafeed from marine microalgae can be an environmentally sustainable replacement for fishmeal if high-value metabolites are coproduced in a biorefinery.