Abstract: To evaluate the influence of light intensity and temperature variation on photosynthetic performance, oxidative defense capacity, and intracellular organic matter accumulation in Ulothrix sp., algal cultures were subjected to gradients comprising six levels of irradiance and six temperature regimes for a 10-day period. Biomass production, chlorophyll fluorescence induction kinetics, antioxidant enzyme activities, and cellular nutrient composition were quantified. Results indicated that maximum growth occurred at an irradiance of 10 000 lx and a temperature of 25 °C. Both excessively high and excessively low light intensities induced pronounced increases in catalase (CAT) and superoxide dismutase (SOD) activities together with elevated malondialdehyde (MDA) concentrations, indicating enhanced oxidative stress. Under these unfavorable light conditions, pigment composition underwent regulatory adjustment. Notably, low irradiance markedly stimulated lipid content, reaching 38.1% of dry weight. At 10 °C and 15 °C, algal growth remained relatively stable, accompanied by increased antioxidant enzyme activities and significant elevations in intracellular polysaccharide and protein concentrations. In contrast, exposure to 35 °C severely inhibited algal growth and photosynthetic activity, although lipid content increased. Overall, Ulothrix sp. cells exhibited high polysaccharide and lipid levels but comparatively low protein content. Under environmental stress, physiological stability was maintained through activation of oxidative stress defense mechanisms and reorganization of intracellular nutrient allocation, facilitating metabolic acclimation to adverse environmental conditions.