OBJECTIVE Alternaria alternata is a major contaminant of wine grapes, meaning a health risk for wine consumers due to the accumulation of toxic metabolites. To develop a successful biofungicide, the effectiveness of epiphytic wine grape yeasts against A. alternata growth and toxin production was assessed in vitro under temperature and aW conditions that simulate those present in the field. RESULTS The effect of 14 antagonistic yeasts was evaluated on growth and alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA) production by three A. alternata strains in a synthetic medium with composition similar to grape (SN) at three temperatures (15, 25 and 30°C). All Metschnikowia sp. yeast strains evaluated completely prevented A. alternata growth and mycotoxin production at all temperatures in SN medium. Meanwhile, the growth inhibition exerted by Starmerella bacillaris yeast strains was higher at 30°C, followed by 25 and 15°C, being able to show a stimulating or inhibiting effect. Hanseniaspora uvarum yeast strains showed a growth promoting activity higher at 15°C, followed by 25 and 30°C. Even at conditions where A. alternata growth was stimulated by the S. bacillaris and H. uvarum yeasts, high inhibitions of mycotoxin production (AOH, AME and TA) were observed, indicating a complex interaction between growth and mycotoxin production. CONCLUSIONS There is a significant influence of temperature on the effectiveness of biocontrol against A. alternata growth and mycotoxin production. Metschnikowia sp. strains are good candidates to compose a biofungicide against A. alternata. CONCLUSIONS Among the different antagonistic yeasts evaluated, only Metschnikowia sp. strains were equally effective reducing A. alternata growth and mycotoxin at different temperatures underlining the importance of considering environmental factors in the selection of the antagonists.