Abstract The impact of five fungicides (prochloraz, propioconazole, epoxiconazole, tebuconazole and azoxystrobin, 0.5–50 μg g−1) on growth of two Fusarium graminearum was evaluated in relation to water activity (aW, 0.99, 0.97, 0.95) and temperature (15°C and 25°C) on wheat-based media (in vitro). All fungicides reduce growth rates when compared to the control, and this reduction increased as the fungicide concentration increased. In general, none of the isolates was able to grow in the presence of any fungicide treatments at concentrations >15 μg ml−1, regardless of the aW/temperature regime. The same fungicides were used in the second study on wheat grain (in situ), in order to evaluate the effect of two concentration (0.5, 5 μg ml−1), three aW levels (0.995, 0.99 and 0.97) and two temperatures (15°C and 25°C) and their interaction on growth rate and deoxynivalenol (DON) production by F. graminearum. All fungicides showed inhibition of growth at both concentrations in most conditions. The fungicides tested were less effective on grain in controlling growth than in in vitro studies. It was noticeable that at 15°C and 0.99 and 0.97 aW, two of the fungicides (tebuconazole and epoxiconazole) showed growth stimulation. All fungicides showed DON stimulation or reduction in at least one of the conditions assayed. Our results show that stimulation or reduction in DON production in the presence of fungicides is influenced by complex interactions between aW, temperature, fungicide concentration and time of incubation in both strains of F. graminearum studied. Such information is critical for effective fungicide control of Fusarium head blight of wheat.