Although biogas is a renewable energy source alternative to natural gas, it contains approximately 40 vol% CO2 and, hence, a low calorific value. The sequestration of CO2 from biogas is, therefore, essential before its widespread use. As CO2 can be easily solubilized as carbonate and bicarbonate in alkaline water, in this study, we isolated and characterized alkaliphilic wild microalgae that grow under high-level CO2 conditions and evaluated their application potential in CO2-removal from biogas. For this purpose, freshwater samples were enriched with 10 vol% CO2 and an alkaline culture medium (pH 9.0), wherein almost free CO2 was converted to carbonate and bicarbonate to yield alkaliphilic and high-level CO2-tolerant microalgae. Ten microalgal strains of Micractinium, Chlorella, Scenedesmus/Tetradesmus, or Desmodesmus spp. were isolated, some of which demonstrated good growth even under conditions of >pH 10 and >30 vol% CO2. All algal strains grew well through fixing biogas-derived CO2 in a vial-scale biogas upgrading experiment, which reduced the CO2 level in biogas to an undetectable level. These strains yielded antioxidant carotenoids, including lutein, astaxanthin, zeaxanthin, and β-carotene, particularly rich in lutein (up to 7.3 mg/g dry cells). In addition, these strains contained essential amino acids, accounting for 42.9 mol% of the total amino acids on average, and they were rich in unsaturated fatty acids (comprising 62.2 wt% of total fatty acids). The present study identified strains that can contribute to biogas upgrading technology, and the present findings suggest that their biomass can serve as useful raw material across the food, nutraceutical, and feed industries.