This study was aimed to optimize the culture conditions for the growth of Acinetobacter calcoaceticus SE370 to produce bioactive substances for plant growth promotion. A basal medium containing 0.3% yeast extract, 0.3% Na₂HPO₄, 0.05% MgCl₂, and pH of 7.0 was used as the control. Sources of carbon (glucose, fructose, sucrose, and mannitol), and nitrogen (peptone, tryptone, NH₄Cl, KNO₃, and yeast extract) and different culture temperatures (25°C, 30°C, 35°C and 38°C) were used for the control medium for A. calcoaceticus SE370 culture optimization. The rate of cell growth was higher for the optimal medium containing 1.5% glucose and 1% tryptone in a basal medium at 35°C when compared to that for the control. The production of malic, citric, succinic, and gibberellic acids was high by A. calcoaceticus SE370 strain cultured in the optimal medium. Further, an enhanced level of phosphate solubilization was noted in A. calcoaceticus SE370 cultured in the optimal medium. The growth of mustard and Chinese cabbage seedlings significantly increased by using A. calcoaceticus SE370 cultured in optimal medium, resulting in increased shoot and root lengths, fresh weight, and chlorophyll content. Thus, an optimal medium composition for A. calcoaceticus SE370 culture enhanced the production of plant growth promoting substances, subsequently accentuating plant growth.