Gilaburu (Viburnum opulus L.) is an important fruit that has been studied in recent years due to its phytochemicals and health benefits. In this study, traditionally produced vinegar made from gilaburu fruit (C-GV) was evaluated. Vinegar with higher levels of bioactive components optimized by response surface methodology (RSM) was also produced using ultrasound (UT-GV). The maximum optimization result for the bioactive components was achieved at 14 min and 61.2 amplitude. The effectiveness of thermal pasteurization (P-GV) on gilaburu vinegar was evaluated. An increase was detected for every organic acid with ultrasound treatment. In the UT-GV and C-GV samples, arabinose was present, which is useful for stimulating the immune system. Gilaburu vinegar samples contained 29–31 volatile compounds. The smallest amount of volatile compounds was found in P-GV (1280.9 µg/kg), and the largest amounts of volatile compounds were found in C-GV (1566.9 µg/kg) and UT-GV (1244.10 µg/kg). In the UT-GV sample, Fe was increased, but Ca, K, Mg, and Mn were decreased. A total of 15 polyphenols were detected in C-GV, P-GV, and UT-GV samples, and gallic acid was the most common. A total of 17 free amino acids were detected in gilaburu vinegar samples. Ultrasound provided enrichment in total phenolic compounds and total free amino acids. All three vinegar samples had good antimicrobial activity against pathogens. The efficacy of C-GV, P-GV, and UT-GV samples against colon and stomach cancer was determined, but there were no significant differences between them. As a result, ultrasound treatment is notable due to its antimicrobial and anticancer activity, especially for the enrichment of phenolic compounds and amino acids in gilaburu vinegar.