Ancient Natural Fertilizer (Jivamrit) is a traditional liquid natural mixture that undergoes fermentation and is frequently applied to enhance the soil microbiome. It is a great source of biomass with macro and micronutrients needed by crops, as well as increasing the organic carbon in the soil. With a thorough nutritional analysis that included bacterial isolation, biochemical tests, and bacterial identification by 16S rRNA sequencing, and nutrient analysis of Jivamrit, the current study concentrated on the isolation along with characterization of the major culturable bacteria from Jivamrit prepared using Gir cow native to Gujarat, India. Jivamrit yielded a total of fifteen major isolates, which were utilized for biochemical analysis and morphological evaluation. The biochemical analysis (catalase, indole synthesis, methyl red, VP test, urease, citrate, sucrose fermentation) and plant growth-promoting assays were performed on all isolated bacteria. The chemical analysis of Jivamrit revealed that it contains large amounts of macro and micronutrients and, importantly, contains Azotobacter, Zinc solubilizing bacteria, and total potash mobilizing bacteria. Molecular characterization of bacterial isolates was performed by amplifying the 16S rRNA gene using bacterial universal primers. PCR-amplified product was sequenced and was carried out, and its analysis revealed that the majority of isolates belong to the Bacillus and Priestia genera. A phylogenetic and evolutionary analysis imparts that the majority of isolates belonged to a different Bacillus species, while JW6 and JW9 belonged to Priestia megaterium. Thus, the present study revealed that major cultivable Bacillus and Priestia bacterial genus are present in Jivamrit. A literature survey revealed that the identified species immensely contribute to increasing soil fertility, plant growth, and yield. Therefore, Jivamrit acts as an alternative to chemical manure and works as a natural fertilizer, which minimises the hazardous effects of chemical farming practices on human well-being and the surroundings.