Microalgae as one of the key components of food chains in aquatic environments are promising biological models for investigating gamma irradiation effects on eukaryotic organisms. Understanding resistance mechanisms in these organisms as simple models might illuminate how gamma irradiation resistance improves in these algae and even more complex organisms. The present study aimed to investigate the effects of ionizing irradiation on Chlorella vulgaris as a stress-tolerant microalga and to find how the eukaryotic cells would tolerate such stressful conditions. The physiological responses and biochemical alterations of C. vulgaris were analyzed at three different time points (0, 16, and 48 h) after 600 Gy gamma irradiation. Compared to the control, gamma-irradiated algae had slower growth rate with significantly longer lag phase, less chlorophyll and protein contents at time 0, which were compensated and recovered during the next 48 h. The results also showed spontaneous H2O2 burst accompanied by a higher rate of lipid peroxidation and electrolyte leakage, a rapid increase of catalase activity and more ferric reducing antioxidant power immediately after irradiation. During a 48-h period, most alterations stabilized. Raising trends were observed in the carotenoid contents, the ratios of carbohydrates, amide I and amide II to fatty acids. Principal component analysis and hierarchical clustering suggest two possible distinct mechanisms: a “quick” one including spontaneous responses by boosting H2O2, amplifying enzymatic antioxidant systems, and increasing compatible solutes like proline and a “delayed” responsive strategy including the increase of soluble carbohydrates, carotenoids, and stress-related proteins triggered several hours after irradiation.