Brettanomyces bruxellensis is a yeast that causes spoilage in red wines due to its ability to produce volatile phenols, compounds associated with major sensory defects. Specific monitoring of low populations of this species in complex ecosystems such as wine during fermentation or aging often relies on plating onto selective media supplemented with cycloheximide. However, the variability of B. bruxellensis sensitivity to this antibiotic needs to be better characterized. A collection of 175 B. bruxellensis strains was, thus, grown on YPD medium supplemented with increasing concentrations of cycloheximide (0 to 0.5 g.L⁻¹), and yeast development was monitored for 20 days by image analysis. This study revealed significant inter-strain variability, with some strains showing very late or even no growth at high cycloheximide concentrations. The cycloheximide inhibitory effect was also dose- and population-dependent. In addition, colony size was frequently reduced at high doses. Additional tests were conducted on a subset of strains grown in wines with either low pH or high alcohol content or containing sulfur dioxide and then plated in the presence of increasing concentrations of cycloheximide. This revealed a cumulative effect of wine and cycloheximide stresses that resulted in an even higher delay in yeast detection. The results confirm the huge phenotypic diversity of the species and highlight the need to adapt the plates’ incubation time, particularly when the selectivity and the doses of cycloheximide needed are high (samples taken in pre-fermentation phases or during fermentation) or in case of stressful wine analysis, to minimize the risk of false negatives.