The demand for citric acid (CA) as a component of food products, pharmaceuticals, and cosmetics is increasing yearly. The use of adapted micro-organisms that convert naturally occurring carbohydrates into organic acids makes it possible to increase annual CA production significantly. The research aim was to study CA production by the Aspergillus niger strain in the medium based on carbohydrate-containing raw materials as a carbon source. We used a fermentation by A. niger. Starch hydrolysates were chosen as a nutrient medium. To improve the CA production of A. niger, multi-step mutagenesis was performed. This resulted in mutant strain A. niger R5/4, which had the highest acidogenic activity among the samples. The study evaluated the effect of temperature on the productivity of the mutant strains. The quantitative content of citric acid was analyzed at different incubation times (144, 168, and 192 h). The effect of the initial medium pH (4.5, 5.0, and 5.5) on acid formation was also investigated. The strain's optimum temperature, pH, and cultivation time parameters were determined. A three-factor, three-level Box-Benken design (BBD) was used to optimize CA production by A. niger strain R5/4 on a starch-containing medium. When assessing the impact of temperature on CA production, the ideal range was between 29 and 31 °C.