Acetoin is widely used as flavor agent and serves as a precursor for chemical synthesis. Here we focused on identifying the best physiological conditions (initial substrate concentrations, pH, temperature, and agitation) for enhanced acetoin accumulation by Bacillus subtilis 168. The optimal physiological conditions support maximum acetoin accumulation by minimizing byproduct (acetate and butanediol) synthesis and a maximum of 75% enhancement in acetoin yield could be achieved. Additionally, the effect of change in ALS (acetolactate synthase) and ALDC (acetolactate decarboxylase) activities was evaluated on acetoin accumulation. Increasing ALS and ALDC enzyme activities led to efficient utilization of pyruvate towards acetoin accumulation and about 80% enhancement in acetoin accumulation was observed.