Small interfering RNAs (siRNA) have a broad potential as therapeutic agents to reversibly silence any target gene of interest. The clinical application of siRNA requires the use of safe and effective delivery systems. In this study, we investigated the use of non-ionic surfactant vesicles (NISV) for the delivery of siRNA. Different types of NISV formulations were synthesised by microfluidic mixing and then evaluated for their physiochemical properties and cytotoxicity. The ability of the NISV to carry and transfect siRNA targeting green fluorescent protein (GFP) into A549 that stably express GFP (copGFP-A549) was evaluated. Flow cytometry and Western blotting were used to study the GFP expression knockdown and significant knockdown was observed as a result of siRNA delivery to the cells by NISV. This occurred in particular when using tween 85, which was able to achieve more than 70% GFP knockdown. NISV were thus demonstrated to provide a promising and effective platform for therapeutic delivery of siRNA.