Biotransformation of a pharmaceutical precursor, phenylacetylcarbinol (PAC), could be achieved by frozen-thawed Candida tropicalis whole cells (FT-WHC). The treatment of FT-WHC, which contained intracellular pyruvate decarboxylase (PDC) enzyme, using high-power ultrasonication with varying amplitudes were compared with glass bead attrition (GBA) as well as control for the release of PDC. Ultrasonication at 20% amplitude (Ult20) proved to be the most effective, resulting in the highest volumetric and specific PDC activities of 0.210 ± 0.004 U/mL and 0.335 ± 0.033 U/mg protein, respectively. Disrupted FT-WHC using Ult20 exhibited a statistically significant (p ≤ 0.05) higher initial PAC production rate (3.26 ± 0.04 mM). The comparison of three organic phases, namely, vegetable oil (Vg-Oil), Vg-Oil + dipropylene glycol (DPG), and octanol in the two-phase emulsion system for PAC biotransformation revealed the highest statistically significant (p ≤ 0.05) overall PAC concentration of 28.9 ± 0.1 mM in Vg-Oil + DPG system. The novel addition of DPG helped facilitating the partitioning of PAC into aqueous phase, stabilizing specific PDC activity, and specific PAC productivity in combination with ultrasonication treatment.