A Green Technology Comparison framework incorporating a life cycle approach and sustainability metrics has been used to compare the performance, and the environment, health, safety, and life cycle impacts of two synthetic methods used to produce 7-aminocephalosporic acid (7-ACA). The routes under study were a chemical synthetic process and a two-enzyme catalyzed process, both starting from the potassium salt of cephalosporin C. Cradle-to-gate life cycle impact estimations were performed using the Fast Life Cycle Assessment of Synthetic Chemistry (FLASC™) tool and following modular gate-to-gate methodology. The results compare the synthetic efficiency, environment, health, safety, and life cycle metrics for a mature chemical process and a more recent but less developed enzymatic process for making 7-ACA. The chemical process has a higher yield, but a significantly lower reaction mass efficiency and half the mass productivity of the enzymatic process. The chemical process uses more hazardous materials and solvents and requires about 25% more process energy than the enzymatic process. When accounting for the cradle-to-gate environmental life cycle, the chemical process has a larger overall environmental impact, mainly derived from the production of raw materials. In comparison to the enzyme-catalyzed process, the chemical process uses approximately 60% more energy, about 16% more mass (excluding water), has double the greenhouse gas (GHG) impact, and about 30% higher photochemical ozone creation potential (POCP) and acidification impact.