Soaking is an important step in making tempeh. Tempeh fermentation normally involves the natural presence of proteolytic bacteria capable of producing protease enzymes to break down peptide bonds in protein molecules. This study evaluated the protein and amino acid content of Jack bean tempeh (Canavalia ensiformis) soaked in distilled water during natural fermentation for 12, 24, 36, and 48 h. In this study, the crude proteins were determined using the Kjeldahl technique, amino acids were determined from protein hydrolysis, and proteolytic bacteria were enumerated for Total Plate Counts and further identified using Vitek 2.0 Compact System. The results showed that soaked Jack beans have higher protein and amino acid content, with sixteen essential and non-essential amino acids required for human bodies. The protein content of soaked Jack beans varied from 35% at 12 h to 32% at 24 and 36 h and 33% at 48 h. Soaking for 12 h yielded the highest amino acid concentration of 38,000 mg/kg L-glutamate, and the lowest of 14,000 mg/kg L-proline. Seven isolated bacteria showed proteolytic activity on Skim Milk Agar with a clear zone of 3.00 mm to 10.65 mm surrounding the colony. The bacteria identified were Pediococcus pentosaceus, Stenorophomonas maltophilia, Cronobacter sakazakii, and Klebsiella pneumonia ssp. In summary, Lactobacillaceae and Enterobacteriaceae were the predominant bacteria during tempeh fermentation, indicating the synergistic interaction between these microflorae during soaking conditions as part of their survival in this hostile environment.