The effects of cultivation mode, photoperiod regime, indole-3-acetic acid (IAA), and glycerol supplementation on the growth performance and morphology of Chlorella vulgaris were evaluated under autotrophic, mixotrophic, and heterotrophic culture conditions. The experiment was arranged in a completely randomized design consisting of six treatments with three replicates. Cultures were maintained for seven days under continuous light, continuous darkness, or a 12 h light:12 h dark photoperiod, with selected treatments supplemented with 10 mg L⁻¹ IAA and 0.05 mL L⁻¹ glycerol. Cell density, cell diameter, temperature, pH, nitrate, and phosphate concentrations were monitored throughout the cultivation period. Significant differences in growth performance and cell morphology were observed among treatments (p < 0.05). The autotrophic control (KA) produced the highest cell density, reaching 389.73 ± 4.15 × 10⁶ cells mL⁻¹ on day 5, whereas the mixotrophic treatment supplemented with IAA and glycerol (M) exhibited the lowest cell density (144.00 ± 1.50 × 10⁶ cells mL⁻¹). In contrast, the largest mean cell diameter was recorded in treatment M (6.30 ± 0.12 μm), followed by the heterotrophic treatment supplemented with IAA and glycerol (H) (5.95 ± 0.09 μm). Water quality parameters remained within suitable ranges for microalgal growth throughout the experiment. Residual nitrate and phosphate concentrations were generally lower in treatments with higher cell densities, indicating greater nutrient utilization. The results demonstrate that cultivation mode and photoperiod exerted a stronger influence on biomass production than IAA and glycerol supplementation under the conditions tested. Continuous illumination favored cell proliferation, whereas the combination of glycerol and IAA promoted cellular enlargement. These findings provide insight into the trade-off between population growth and cell morphology in C. vulgaris cultivation systems.