Poppy vinegar with functional properties is a fermented product. This study evaluated traditionally produced poppy vinegar. The study was conducted on poppy vinegar to determine the maximum increase in angiotensin converting enzyme (ACE) inhibitory activity %, total phenolic content (TPC), and radical scavenging activity (DPPH) of the vinegar at different combinations of ultrasound treatment duration (2−14 min) and amplitude (40−100%). The optimal parameters obtained using the response surface methodologies (RSM) were the duration of the ultrasound of 5.5 min and the amplitude of the ultrasound at 57%. When the DPPH values, ACE inhibition %, and TPC and DPPH values obtained with the RSM model were compared with the experimental values, the difference was 9.80, 3.0, and 4.6%, respectively, showing good agreement between actual and predicted values. The higher ultrasound intensities and longer treatment times had a significant effect on antioxidant activity. Poppy vinegar samples significantly induced the apoptosis of lung cancer cells, particularly those stored for 6 and 12 months. The amounts of protocatechuic acid, gallic acid, neohesperidin, hydroxybenzoic acid, resveratrol, rutin, transcinnamic acid, quercetin, and flavon in poppy vinegar were determined, which decreased significantly as storage time increased. TPC and TFC were determined to be 90.39 mg of GAE/100 mL and 29.86 mg of TEAC/mL, respectively, and there was no significant change in these bioactive compounds after 6 months of storage. The highest value of ACE inhibitory activity was found at the beginning of the storage period. The present study was the first study to examine the bioactive components, ACE inhibition activity, pro-apoptotic activities, and phenolic composition of traditionally produced ultrasound-treated poppy vinegar during storage. The control of production parameters and the design of ideal poppy vinegar fermentation processes could benefit from this research.