This study reports the results of an evaluation of the techno-economic feasibility of a biorefinery with an annual lactic acid production capacity of 100,000 metric tons using lignocellulosic biomass. Corn stover and miscanthus were considered as model feedstocks, and three different fermentation pathways involving bacteria, fungi, and yeast were compared with respect to their ability to convert biomass feedstocks to lactic acid. Equipment, raw materials, utilities and labor requirements, and lactic acid production costs were estimated. The minimum selling price (at a 10% internal rate of return) per metric ton of lactic acid produced from different feedstocks for lactic acid bacteria, fungi, and yeast-based pathways were in the range of USD 1243–1390, USD 1250–1392, and USD 993–1123, respectively, with lower costs for miscanthus. Lactic acid production using genetically engineered yeast strains can eliminate the need for the simultaneous neutralization and recovery of lactic acid, resulting in lower equipment, chemical, and utility requirements and lower lactic acid production costs. Lactic acid production costs were highly sensitive to the conversion rates of sugars into lactic acid, feedstock cost, production plant size, operation hours, and acid hydrolysis reactor costs. Improvements in process conditions and efficiencies and lower costs of equipment and consumables are necessary to utilize lignocellulosic biomass for lactic acid production at lower costs while remaining cost-competitive with respect to first-generation and petroleum-based feedstocks.