Aims The control of the wine spoilage yeast Brettanomyces bruxellensis using biological methods such as killer toxins (instead of the traditional chemical methods, e.g. SO2) has been the focus of several studies within the last decade. Our previous research demonstrated that the killer toxins CpKT1 and CpKT2 isolated from the wine yeast Candida pyralidae were active and stable under winemaking conditions. In this study, we report the possible mode of action of CpKT1 on B.bruxellensis cells in red grape juice. Methods and Results Brettanomyces bruxellensis cells were exposed to CpKT1 either directly or through co-inoculation with C. pyralidae. This exposure yielded a temporary or permanent decline of the spoilage yeast population depending on the initial cell concentration. Scanning electron microscopy revealed cell surface abrasion while propidium iodide viability staining showed that CpKT1 caused plasma membrane damage on B.bruxellensis cells. Our data show that the exposure to CpKT1 resulted in increased levels of beta-glucan, suggesting a compensatory response of the sensitive cells. Conclusions The toxin CpKT1 causes cell membrane and cell wall damage in B. bruxellensis. Significance and Impact of the Study Candida pyralidae shows potential to be used as a biocontrol agent against B. bruxellensis in grape juice/wine.