The worldwide increase in diabetes entails a rise in associated diseases, with diabetic retinopathy on the forefront of the ocular complications. To overcome the challenges posed by ocular barriers, self-assembled nanocarriers have gathered increasing attention in recent years, with niosomes revealing themselves to be suitable for the delivery of a variety of drugs. This study investigated the mechanical properties of Langmuir monolayers comprising cholesterol, Tween 60, and 1,2-di--octadecenyl-3-trimethylammonium propane (DOTMA), both individually and in binary and ternary systems. The cholesterol monolayer was characterized by an L-shaped isotherm, reflecting two surface aggregation states. Tween 60 exhibited expanded conformation and progressive aggregation, transitioning through a phase change. The addition of cholesterol to Tween 60 resulted in a subtle reduction in surface compressional modulus. The compression isotherms highlighted the stabilizing effect of cholesterol on the monolayer, affecting the film's resistance to compression. The introduction of DOTMA in Tween 60 monolayers revealed concentration-dependent effects, where the compression resistance of the film was proportional to DOTMA concentration. Ternary systems of cholesterol, DOTMA and Tween 60 exhibited unique behavior, with DOTMA enhancing film stability and cholesterol modulating this effect. Temperature and subphase ionic strength variations further exacerbated the effects of DOTMA concentration. Brewster Angle Microscopy confirmed the absence of microdomains in the compressed monolayer, supporting the hypothesis of a monolayer collapse. Overall, the research provided valuable insights into the intricate interactions and mechanical behavior of these surfactant systems and the feasibility of obtaining cationic niosome-based drug delivery.