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Abstract BACKGROUND In several countries, the cactus plant (Opuntia ficus‐indica (L). Mill.) has received renewed attention because of its ecological, socio‐economic and environmental role. In this study, prickly pear vinegar was produced employing two types of acetification processes: surface and submerged culture. Both acetification processes were performed at different temperatures (30, 37, 40 °C) by using two different species of thermotolerant acetic acid bacteria (Acetobacter malorum and Gluconobacter oxydans). Polyphenols and volatile compounds analyzed by ultra‐performance liquid chromatography with diode array detection and stir bar sorptive extraction–gas chromatography–mass spectrometry, respectively, were considered as the main variables to determine the effect of the acetification process on the quality of the vinegar. RESULTS As a result, 15 polyphenols and 70 volatile compounds were identified and quantified in the vinegar samples produced by both acetification processes. The results showed that the surface acetification method led to an increase in the concentration of phenolic components, which was higher than that in the submerged process. However, a significant increase in volatile compounds predominated by esters and acids was observed when submerged culture acetification was employed, whereas alcohols were predominant in surface culture vinegars. Moreover, multivariate statistical analysis showed that the components that mostly contributed to the differentiation between all vinegar samples were the volatile compounds. CONCLUSION It has been proved that prickly pear vinegar could be successfully produced at higher temperatures than usual, by employing thermotolerant bacteria, and that the type of acetification method significantly affects the final quality of the vinegar produced. © 2022 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.