Time-dependent cytosolic and mitochondrial alterations in non-cancerous and cancerous lung cells exposed to 5 Gy ionizing radiation were monitored over five days. Oxidative stress increased more rapidly in cancerous cells, affecting both compartments. In cancerous mitochondria, GSH levels decreased, while cytosolic GSH increased post-radiation. In contrast, non-cancerous mitochondria exhibited a transient GSH increase. Malondialdehyde levels rose in non-cancerous cells but declined in cancerous mitochondria. Basal ATP levels were higher in the cytosol of non-cancerous cells, while mitochondrial ATP remained comparable. Superoxide dismutase and glutathione peroxidase activities increased in non-cancerous cells, whereas catalase decreased in non-cancerous and increased in cancerous cells. Mitochondrial membrane potential, permeability transition pore integrity, and mitochondrial mass were significantly impaired in cancerous cells. Apoptotic markers were absent in non-cancerous cells except for Bax translocation on day 3. In cancerous mitochondria, cytochrome c and apoptosis-inducing factor peaked on days 1 and 5 but dropped on day 3, accompanied by a marked caspase-3 increase. ETC complexes showed significant radiation-induced alterations at protein, activity, and mRNA levels, differing between cell types. These findings highlight that mitochondrial oxidative stress is induced, enzymatic antioxidant defenses are weakened, apoptotic pathways are activated, and ETC complex activities are less responsive to radiation in cancerous cells compared to non-cancerous cells.