The increasing worldwide demand for antimicrobial agents has significantly contributed to the alarming rise of antimicrobial resistance, posing a grave threat to human life. Consequently, there is a pressing need to explore uncharted environments, seeking out novel antimicrobial compounds that display exceptionally efficient capabilities. Hot springs harbor microorganisms possessing remarkable properties, rendering them an invaluable resource for uncovering groundbreaking antimicrobial compounds. In this study, thermophilic bacteria were isolated from Mahallat Hot Spring, Iran. Out of the 30 isolates examined, 3 strains exhibited the most significant antibacterial activities against Escherichia coli and Staphylococcus aureus. Furthermore, the supernatants of the isolated strains exhibited remarkable antibacterial activity, displaying notable resistance to temperatures as high as 75 °C for 30 min. It was determined that the two strains showed high similarity to the Bacillus genus, while strain Kh3 was classified as Saccharomonospora azurea. All three strains exhibited tolerance to NaCl. Bacillus strains demonstrated optimal growth at pH 5 and 40 °C, whereas S. azurea exhibited optimal growth at pH 9 and 45 °C. Accordingly, hot springs present promising natural reservoirs for the isolation of resilient strains possessing antibacterial properties, which can be utilized in disease treatment or within the food industry.