Niosomes are non-ionic surfactant vesicles that has successfully attracted a great deal of attention over the last few decades. The best ratio between main components is the key to creating the optimum formulations with substantial loading capacity. Therefore, this study investigated formulation factors that could affect the entrapment of two compounds with different solubilities, namely Methylene blue (MB) and curcumin (CUR) as model hydrophilic and hydrophobic compounds, respectively. The aim was to propose an optimum formula to encapsulate both compounds as a potential hybrid anticancer therapy. The factors with potential influence on the entrapment efficiency (EE) included the type of the non-ionic surfactants (Span 60 versus Span 65), cholesterol to surfactant ratio, and the concentrations of the co-surfactant (Cremophor RH40). Niosomes were prepared by thin film hydration technique and downsized using probe sonicator. The morphology, vesicle size and polydispersity index (PDI) of the prepared vesicles were inspected. The data showed the superiority of Span 65 over Span 60 in terms of higher encapsulation efficiency for MB and CUR. The results reflected positive impact of cholesterol and Cremophor RH40 concentration on EE of the two compounds. The optimum composition of Span 65:cholesterol:Ccremophor RH40 was at the mol% ratio of 45:45:10, respectively, with EE of 91.19% for CUR alone, 51.70% for MB alone, and 45.32%/84.40% for combined MB/CUR loaded formulations. The vesicle size of the formulations after probe sonication, fall into the range of 100-200 nm, ideal for parenteral delivery to the target sites.