Brettanomyces bruxellensis yeast produces ethyl phenols, which cause wine spoilage and severe economic losses. Ethyl phenols are produced by a class of polyphenols that are present in grapes and musts, called hydrocinnamic acids, which are capable of inhibiting yeasts and bacteria. The viability and intracellular pH changes in B. bruxellensis DSM 7001, in response to extracellular pH, as well as to the presence of an energy source and hydroxycinnamic acids, have been investigated in this paper by means of Fluorescent Ratio Imaging Microscopy (FRIM). The results show that B. bruxellensis DSM 7001 is able to maintain viability and increase its pH gradient with decreasing external pH values, whereas it is not able to maintain a pH gradient at high external pH values (i.e. pH 8) and, as a consequence, dies. The growth inhibitory effects of ferulic and p-coumaric acid do not seem to be caused by a weak-acid inhibition mechanism, since both acids induce a similar, or even higher, intracellular acidification at a high external pH than at a low external pH. The results presented have to be confirmed by using other strains of B. bruxellensins in order to validate the outcomes obtained in this study.