The extended use of niosomes in food, cosmetic and pharmaceutical industries demands novel preparation processes which could enhance nanovesicles production with controlled size and size distribution. Microfluidics (MF) are a novel promising set of preparation methods for this purpose. Studies comparing methods for niosome preparation are still scarce. In this work, niosomes using MF were produced and the effect of membrane compounds concentration and the relation between organic and aqueous ratio (flow rate ratio, FRR) on the resulting mean size and size distribution were studied. Two different cholesterol free formulations were tested using Tween (R) 80 and Span (R) 80 in presence and absence of 1-dodecanol as membrane stabilizer. The effect of FRR showed to be the key parameter in niosomes production, being more critical than membrane compounds concentration and the vesicle membrane composition. Additionally, the same formulations were also produced by two conventional methods, thin film hydration (TFH) and ethanol injection (EI), and results were compared. The size of the niosomes obtained by MF was smaller and more homogeneous than those achieved by TFH and similar to EI. Therefore, MF is confirmed as a novel technology that can facilitate the development and optimization of new nanodelivery systems with controlled size and polydispersity, two essential parameters for successful biotechnological applications. (C) 2020 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.