Mixed fermentation is one of the methods used in sour beer production. The process requires initialization of the fermentation step by well-planned addition of brewing yeast and lactic acid bacteria to slightly hopped wort. The final product’s properties strictly depend on how the microorganisms are pitched and the initial wort composition. The experiment was performed to evaluate the impact of different initial conditions and pitching methods on the mixed fermentation process and the final product’s characteristics. With the aim of limitation of the number of experiments, the Box–Behnken design was applied. Three independent factors were considered while obtaining the response surface: initial extract, bitterness and order of pitching. The final product’s properties: ethanol and lactic acid concentration, appeared to depend strictly on initial conditions and pitching order. Several important observations have been made; for example, it appeared that the presence of LAB does not significantly impact the final ethanol concentration. Optimal conditions for obtaining the maximum or minimum of each quality were calculated using Matlab. Obtained results might improve the sour beer production process while shortening the duration and reducing the usage of ingredients.