The aim of the present study was to isolate, identify, characterize, and screen the enzymatic activities of thermophilic bacteria from Vajreshwari hot spring, Maharashtra, India. The three isolates were characterized by morphological, biochemical, and microscopic characteristics. The optimum temperature for the growth was 50 °C, and the pH was 8. The sequencing of the 16S rRNA of the isolates followed by a BLAST search revealed that the three strains were Pseudoxanthomonas taiwanensis strain (NFC7-A), Anoxybacillus salavatliensis strain (Y3MT21), and Bacillus licheniformis strain (HAZ6-17). Pseudoxanthomonas taiwanensis and Anoxybacillus salavatliensis were reported for the first time from the study area, and both strains showed positive activity for amylase while all three bacteria showed positive activity for oxidase and catalase. The results showed that the isolated bacteria from hot springs could be a great source of biotechnologically important thermostable enzymes.