The large-scale continuous production of niosomes remains challenging. The inherent drawbacks of batch processes such as large particle polydispersity and reduced batch-to-batch reproducibility are here overcome by using commercially available microfluidic reactors. Compared to the traditional batch-based film hydration method, herein, we demonstrate that it is possible to carry out the homogeneous, large-scale (up to 120 mg/min) production of niosomes using two different synthesis techniques (the thin film hydration method and the emulsification technique). Niosomes particle size can be controlled depending on the need by varying the synthesis temperature. The high cytocompatibility of the resulting niosomes was also demonstrated in this work on three different somatic cell lines. For the first time, the structure of the niosome multilamellar shell was also elucidated using high-resolution transmission electron microscopy (HR-STEM) as well as their colloidal stability over time (6 weeks) under different storage conditions. The morphology of cryo-protected or as-made niosomes was also evaluated by HR-STEM after freeze-drying. Finally, the dual ability of those synthetic, nonionic, surfactant-based vesicles to carry both hydrophilic and hydrophobic molecules was also here demonstrated by using laser scanning confocal microscopy.