The current study used HEK293 and HeLa cell lines to compare liposomes and niosomes as nanocarriers for gene transfer. After preparing and characterizing liposomal and niosomal formulations containing GFP-encoding plasmid DNA, the effectiveness and cytotoxicity of transfection were evaluated in vitro. According to ANOVA and post hoc analysis, liposomes demonstrated a considerably greater transfection efficiency than niosomes in both cell lines (p 0.05). These results show that niosomes provide a safer substitute with decreased cytotoxicity, indicating a trade-off between efficiency and safety, even though liposomes perform better at delivering genes. Therefore, the study offers a comparison framework for choosing appropriate nanocarriers for upcoming gene therapy applications.