(1) Background: Hedera nepalensis (Araliaceae) is a recognized medicinal plant founded in Asia that has been reported to work in antioxidant, antifungal, antimicrobial, and antitumor capacities. (2) Methods: The subcritical fluid extraction of saponin from Hedera nepalensis leaves and the optimum of the extraction process based on yield of saponin contents (by calculating the hederacoside C contents in dried Hedera nepalensis leaves) are examined by response surface methodology (RSM). Furthermore, the antimicrobial activity of the extract is tested for potential drug applications in the future. (3) Results: Based upon RSM data, the following parameters are optimal: extraction time of 3 min, extraction temperature of 150 °C, and a sample/solvent ratio of 1:55 g/mL. Under such circumstances, the achieved yield of saponin is 1.879%. Moreover, the extracts inhibit the growth of some bacterial strains (Streptococcus pneumoniae, Streptococcus pyogenes, Haemophilus influenza) at a moderate to strong level with inhibition zone diameter values ranging from 12.63 to 19.50 mm. (4) Conclusions: The development of such a model provides a robust experimental process for optimizing the extraction factors of saponin contents from Hedera nepalensis extract using subcritical fluid extraction and RSM. Moreover, the current work reveals that saponin extracts of Hedera nepalensis leaves exhibit a potential antimicrobial activity, which can be used as scientific evidence for further study.