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This study aimed to screen alpha-amylase producing microorganisms from honey as a low water activity medium, a suitable source for selecting stable and cost-beneficial bacterial enzyme production systems. Plackett–Burman method was used to select twelve effective factors including pH, inoculum size, temperature, time, corn starch, KH2PO4, peptone, MgSO4, CaCl2, NaCl, glycerin, and yeast extract concentrations on bacterial alpha-amylases production yield. The Box–Behnken method was utilized to optimize the level of selected significant factors. The stability of bacterial alpha-amylases was also determined in low pH and high-temperature conditions. In addition, in silico study was used to create the alpha-amylase structure and study the stability in high-temperature and low water available condition. Among all isolated and characterized microorganisms, Bacillus megaterium produced the highest amount of alpha-amylases. The in silico data showed the enzyme 3D structure similarity to alpha-amylase from Halothermothrix orenii and highly negative charge amino acids on its surface caused the enzyme activity and stability in low water conditions. Based on Box–Behnken results, the temperature 35 °C, pH 6 and starch 40 g/l were determined as the optimum level of significant factors to achieve the highest alpha-amylases unit (101.44 U/ml). This bacterial alpha-amylases enzyme showed stability at pH 5 and a range of temperatures from 40 to 60 °C that indicates this enzyme may possess the potential for using in industrial processes. [ABSTRACT FROM AUTHOR]