Niosomes are known as non-ionic surfactant-based vesicles and have been used quite a lot for medical applications as a novel drug delivery system in recent years. Herein we describe, the preparation and characterization of niosomes containing oleander (Nerium oleander) (ONs) extract as the herbal compound. In this context, niosomal formulations of two different extracts obtained from oleander roots were prepared by a thin film hydration technique using Tween 60 (non-ionic surfactant) with a cholesterol mixture at 1:1 M ratio. These vesicular structures were characterised by various techniques such as atomic force microscopy, zeta potential and dynamic light scattering size measurements. ONs vesicles are less than 100 nm in size with a good physical stability more than 50 days. Initially, total phenolic and flavonoid contents and antioxidant activities of the extracts were investigated. The methanol extract of oleander roots (MOE) showed higher polyphenolic content and exhibits a better antioxidant activity in compared to the hydro-methanol (20% methanol) extract (MOWE). Total phenolic contents in the MOE and MOWE were calculated as 64.51 +/- 0.945 mu g/mg and 65.05 +/- 0.37 mu g/mg gallic acid equivalents, respectively. Encapsulation efficiencies of the vesicles were found as 16.2% for MON (contain MOE) and 13.24% for MWON (contain MOWE). The significant linear correlation Was confirmed between the antioxidant activity and total phenolic content of extracts as well as ONs. Cell based cytotoxic activities of methanol extract and MON formulations were also assessed via KIT assay using Hela and A549 cell lines. (C) 2016 Elsevier B.V. All rights reserved.