Glucocorticoids in aquatic systems originating from natural excretion and medical use may pose a risk to fish. Here, we analyzed physiological and transcriptional effects of clobetasol propionate (CLO), cortisol and cortisone in zebrafish embryos as single compounds and binary mixtures. CLO and cortisol, but not cortisone showed a concentration-dependent decrease in muscle contraction, increase in heart rate, and accelerated hatching. CLO also induced immobilization and edema at high concentrations. Transcription analysis covering up to 26 genes showed that mostly genes related to glucose metabolism, immune system and development were differentially expressed at 91 ng/L and higher. CLO showed stronger effects on immune system genes than cortisol, which was characterized by upregulation of fkbp5, irg1l, gilz, and socs3, and development genes, matrix metalloproteinases mmp-9 and mmp-13, while cortisol led to stronger upregulation of the gluconeogenesis genes g6pca and pepck1. CLO also induced genes regulating the circadian rhythm, nr1d1 and per1a. In contrast, cortisone led to down-regulation of vitellogenin. Binary mixtures of cortisol and CLO mostly showed a similar activity as CLO alone on physiological and transcriptional end points but additive effects in heart rate and pepck1 upregulation, which indicates that mixtures of glucocorticoids may be of concern for developing fish.