Alcohol is toxic to neurons and can trigger alcohol-related brain damage, neuronal loss, and cognitive decline.  Neuronal cells may be vulnerable to alcohol toxicity and damage from oxidative stress after differentiation. To consider this further, the toxicity of alcohol to undifferentiated SH-SY5Y cells was compared with that of cells that had been acutely differentiated. Cells were exposed to alcohol over a concentration range of 0-200 mM for up to 24 hours and alcohol effects on cell viability were evaluated by MTT and LDH assays. Effects on mitochondrial morphology were examined via transmission electron microscopy, and mitochondrial functionality was examined using measurements of ATP and the production of reactive oxygen species (ROS). Alcohol reduced cell viability and depleted ATP levels in a concentration and exposure duration-dependent manner, with undifferentiated cells more vulnerable to toxicity (viability threshold concentration of ≥20 mM for 6 h, p < 0.001 for undifferentiated cells and p < 0.0001 for differentiated cells). Alcohol exposure resulted in significant neurite retraction (from ≥50 mM for 6 h, p < 0.001), altered mitochondrial morphology, and increased the levels of ROS in proportion to alcohol concentration from 10 mM (p < 0.0001); these peaked after 3 and 6 h exposures and were significantly higher in differentiated cells. Protein carbonyl content (PCC) lagged ROS production and peaked after 12 and 24 h, increasing in proportion to alcohol concentration (from 10 mM, p < 0.0001), with higher levels in differentiated cells. Carbonylated proteins were characterised by their denatured molecular weights and overlapped with those from adult post-mortem brain tissue, with levels of PCC higher in alcoholic subjects than matched controls (p < 0.01). Hence, alcohol can potentially trigger cell and tissue damage from oxidative stress and the accumulation of oxidatively damaged proteins.