Hot springs are known to be rich in diverse microorganisms, including thermophilic bacteria capable of producing industrially important enzymes. This study aimed to isolate, purify, and identify thermophilic bacteria from three hot springs located in the southern region of Saudi Arabia, namely Al-Ma'a Al-Har, Al-Aredhah, and Al-Ahsarai hot spring. The isolated bacterial isolates were screened for amylase enzyme production, and the most potent isolate, Cytobacillus firmus OQ834432 isolate OHA8, was selected for large-scale production of the amylase enzyme. The enzyme was then purified using salt precipitation, Sephacryl S-200 chromatography, and DEAE-Sepharose column chromatography. A total of twenty-one bacterial species were identified, with Cytobacillus firmus exhibiting the highest amylase activity. The optimal conditions for the amylase enzyme activity were found to be at 50℃ and pH 5.0, with a 153% enhancement in activity in the presence of magnesium ions. The enzyme's activity was inhibited by EDTA and was found to be active with starch and amylopectin at 100% and 110.3%, respectively. In conclusion, this study demonstrates the potential of thermophilic bacteria from hot springs to produce industrially important enzymes and provides insights into the optimal conditions for enzyme production and activity. These findings have significant implications for the development of biotechnological applications utilizing thermophilic bacteria in various industrial sectors.