In fungal cells, the endoplasmic reticulum (ER) harbors several of the enzymes involved in the biosynthesis of ergosterol, an essential membrane component, making this organelle the site of action of antifungal azole drugs, used as a first-line treatment for fungal infections. This highlights the need for specific fluorescent labeling of this organelle in cells of pathogenic fungi. Here we report on the development and evaluation of a collection of fluorescent ER trackers in a panel of Candida, considered the most frequently encountered pathogen in fungal infections. These trackers enabled imaging of the ER in live fungal cells. Organelle specificity was associated with the expression of the target enzyme of antifungal azoles that resides in the ER; specific ER labeling was not observed in mutant cells lacking this enzyme. Labeling of live Candida cells with a combination of a mitotracker and one of the novel fungal ER trackers revealed sites of contact between the ER and mitochondria. These fungal ER trackers therefore offer unique molecular tools for the study of the ER and its interactions with other organelles in live cells of pathogenic fungi.