Background/Objectives: The aim of this work is to investigate how corneal oedema influences the thickness of several corneal layers and the refractive index (ncornea), therefore clarifying its effect on corneal structure and endothelial function.Subjects/Methods: A laboratory study was conducted with thirty-four porcine eyes, categorised into three groups: Control (non-oedematous), 24-h oedema, and 48-h oedema. The thickness of corneal layers—epithelium (ET), Bowman’s membrane (BMT), stroma (ST), Descemet’s membrane (DMT), and endothelium (EnT)—was assessed histologically. Endothelial morphology was evaluated using specular microscopy, and corneal measurements were obtained with an Abbe refractometer. Statistical analyses comprised the Kruskal-Wallis test, Spearman correlations, Wilcoxon signed-rank tests, and multiple regression models.Results Control (1.36475 ± 0.0044); 24-h (1.36025 ± 0.0097); 48-h (1.3485 ± 0.0040; p = 0.001); ncornea significantly dropped as oedema progressed. BMT, ST, and EnT were clearly thickened; ST grew by 85% after 48 hours. ncornea revealed a substantial negative correlation with BMT (ρ = -0.491, p = 0.0002) and ST (ρ = -0.684, p = 0.002), but the maximum endothelial cell area (AreaMAX) was favourably associated (ρ = 0.502, p = 0.039). The Wilcoxon test confirmed, at p = 0.001, that ncornea was routinely less than Gullstrand's theoretical value, 1.3771, in all groups.Conclusions Corneal oedema significantly thickens corneal layers and lowers ncornea, therefore affecting optical clarity and measurement accuracy. These findings challenge the idea that ophthalmic equipment should have a fixed corneal refractive index (1.3771), emphasising the need for dynamic index modifications to improve surgical and diagnostic accuracy.