Yeasts of the genus Candida are a major cause of morbidity and mortality in immunocompromised patients. Despite new insights in recent years, the pathogenesis of Candida infection is still incompletely understood. Previous studies have suggested that gliotoxin, a secondary fungal metabolite with well-known immunosuppressive effects, is produced by various species of the genus Candida, and a possible role of gliotoxin as a virulence factor of C. albicans has also been discussed. However, until now, no definitive evidence has been provided that members of the genus Candida are able to produce gliotoxin. To clarify this question, we tested a total of 100 clinical isolates of C. albicans, C. glabrata, C. tropicalis, C. krusei and C. parapsilosis for gliotoxin production using a highly sensitive HPLC protocol, and, for selected isolates, confirmed our findings by tandem MS. This approach did not detect intracellular or extracellular gliotoxin production by any of the isolates examined, although various culture conditions were applied. Therefore, in contrast to previous studies, our data strongly suggest that at least the Candida species investigated in this study are not able to produce the secondary metabolite gliotoxin.