Vascular endothelial growth factor (VEGF) plays a central role in retinal neovascular diseases, and anti-VEGF agents such as ranibizumab and aflibercept are widely used to control these conditions. This study aimed to compare the cytotoxic and autophagic effects of ranibizumab and aflibercept on human retinal pigment epithelium (ARPE-19) cells in vitro. ARPE-19 cells were treated with incremental doses of each drug (1.25× to 40× the clinical concentration). Cell viability was assessed using the MTT assay, and mRNA expressions of autophagy-related genes Beclin1 and ATG4 were quantified by qRT-PCR. Ranibizumab significantly reduced cell viability at 20× and 40× doses compared with the control, whereas aflibercept did not show notable cytotoxicity even at the highest tested concentrations. Both drugs influenced autophagy pathways in different ways: ranibizumab caused less inhibition of Beclin1 expression than aflibercept (p < 0.05), while both agents induced a non-significant increase in ATG4 expression. These results suggest that aflibercept may have a lower cytotoxic potential at supratherapeutic concentrations and that both drugs can modulate autophagic activity in retinal pigment epithelium cells. Understanding these cellular effects may help optimize the safety and long-term application of anti-VEGF therapy in retinal diseases.