This study aims to investigate the morphological and biochemical characteristics of thermophilic bacteria isolated from thermophilic biotopes in the Al-Jouf region of Saudi Arabia for the first time. Additionally, the production of thermostable bacterial enzymes (proteases, amylases, cellulases, gelatinases, and lipases) was investigated. Six main bacterial species were identified via 16S rRNA sequencing and phylogenetic analysis. Twenty water and soil samples were collected from several thermophilic sites in the Dumat Al-Jandal and Al-Qurayyat cities. In total, 28 bacterial strains were isolated and biochemically characterized. Most isolated strains showed high protease and amylase production. 46 and 28% of isolated strains showed the production of cellulase and gelatinase, respectively. However, lipase production was not observed in any isolated bacteria. Four Bacillus licheniformis and two Bacillus subtilis strains exhibited high GC content (55%). Our findings suggest thermophilic bacteria as a source of thermostable enzymes for pharmaceutical and industrial applications.