Quambalaria cyanescens was positively identified on the surface of unripened grapes by physiological and molecular techniques; this is the first work describing the isolation of this fungus in European grapes and all of them produced an exocellular β-glucosidase. Once isolates with higher activity were selected, the enzyme has been qualitatively and quantitatively determined and characterized, by spectrophotometric methods, based on its biotechnological properties Exocellular β-glucosidase produced by Q. cyanescens is inhibited by glucose but is an ethanol-tolerant enzyme and its activity is independent of the pH value. Divalent cations, mainly Ca2+, highly increase enzymatic activity. This is the first work describing the isolation, identification and characterization of Q. cyanescens in grapes. Although there is growing interest in the search for new fungal β-glucosidases, the vast majority of studies were performed with imperfect fungi, and there are scant reports that deal with basidiomycetous fungi, not only for biotechnological purposes but also for physiological research.