Vitamin K-2 (VK2) is a microbial product that has several applications in the pharmaceutical sector, and its yield is very important for commercial processes. This study analyzed the effects of a single surfactant, with or without ultrasound treatment, on the metabolic synthesis of VK2 by Flavobacterium sp. M1-14. The surfactant combined with ultrasound altered the membrane permeability of Flavobacterium sp. M1-14, as shown by scanning electron microscopy and flow cytometry (FCM). The optimum conditions for VK2 production were as follows: amplitude 80% of total power (130 W), sonication time of 40 s, and continuous repetitive ultrasound treatment repeated three times, and the yield of extracellular VK2 reached 30 mg/L when 1% polyoxyethyleneoleyl (POE)was added to the medium. Scanning electron microscopy studies revealed that the mechanism by which the surfactant combined with ultrasound altered the membrane permeability involved both cell membrane pore formation through stable cavitation and solubilization of the membrane. Based on the double-staining results, we concluded that combined ultrasound with surfactant treatment could promote esterase activity, enhance cell membrane permeability, and reduce the damaging effects of surfactant alone applied to the cells.