Smoking remains a significant health problem in patients with type 2 diabetes mellitus. This study compared intracellular Ca²⁺ ([Ca²⁺]_i) in microglia, neurons, and astrocytes in the presence of high glucose (HG) and nicotine and evaluated the effects of Lavandula angustifolia Mill. essential oil (LEO) on this process. [Ca²⁺]_i concentrations were measured by monitoring the fluorescence of Fura-2 acetoxymethyl ester. Treatment with HG and nicotine significantly increased [Ca²⁺]_i in both microglia and neurons through Ca²⁺ influx from extracellular sources. This increased Ca²⁺ influx in microglia, however, was significantly reduced by LEO, an effect partially inhibited by the Na⁺/Ca²⁺ exchanger (NCX) inhibitor Ni²⁺. Ca²⁺ influx in neuron-like cells pretreated with HG plus nicotine was also significantly decreased by LEO, an effect partially inhibited by the L-type Ca²⁺ channel blocker nifedipine and the T-type Ca²⁺ channel blocker mibefradil. LEO or a two-fold increase in the applied number of astrocytes attenuated Ca²⁺ influx caused by high glucose and nicotine in the mixed cells of the microglia, neuron-like cells and astrocytes. These findings suggest that LEO can regulate HG and nicotine-induced Ca²⁺ influx into microglia and neurons through two distinct mechanisms.