This study explored the feasibility of thermosonication (TS)-prestressed inoculum with different fermentation patterns for regulating microbial (post)-fermentation acidification kinetics. Through a Box-Behnken design, stimulative (20 min, 400 W, 33 kHz, 25 °C) and inhibitive (10 min, 600 W, 33 kHz, 20 °C) effects on the acidification capability of Lactobacillus plantarum A3 were achieved without observing greatly activated/inactivated strains growth, further confirmed by lactose fermentation performed by Streptococcus thermophilus and Lactobacillus bulgaricus. Lactic acid was the major contributing factor responsible for TS-induced acidification modifications corresponding to the potential fluctuations of CoA biosynthesis, fatty acid degradation and chain elongation pathways to TS prestress. Microscopy observations and quantitative extracellular substance assays showed palpable stress disturbance on microbes, but causing insignificant effects on product characteristics. This investigation demonstrated the potential of controlled sonication prestress strategies to achieve dual engineering effects on microbial metabolic behavior, for alleviating post-acidification problem or enhancing process efficiencies.