The current study evaluates the impact of substrate to inoculum (S/I) ratio on the quality and quantity of volatile fatty acids (VFAs) produced during food waste (FW) fermentation. Four S/I ratios (1, 2, 3, and 4, on a volatile solid (VS) basis) were experimented in batch mode at 37 degrees C for 9 days. According to the obtained data, acidification yield (eta a) increased from 39.93 to 67.38 mg COD/mg COD as the S/I ratio increased from 1 to 3, but a higher ratio (S/I: 4) led to process inhibition. The highest total VFA production (49.32 g/L) was achieved at S/I: 3, followed by S/I: 2, 1, and 4 (39.07, 27.08, and 27.39 g/L, respectively). VFA composition analysis showed that increasing the S/I ratio shifted the profile from butyric to acetic acid. Maximum hydrogen yields for S/I ratios of 1, 2, 3, and 4 were 56.32, 69.10, 59.82, and 33.46 mL H2/g VSadded, respectively. S/I: 3 also demonstrated significant hydrolysis yield, high VS destruction, strong buffering capacity, and stable hydrogen co-production. Monitoring NADH fluctuations revealed enhanced process stability at S/I: 3. These findings suggest that optimizing the S/I ratio can significantly enhance bio-based VFA production from FW. Acidogenic fermentation thus presents an environmentally friendly and sustainable platform for converting waste into valuable products.