Abstract Background Olorofim is a new antifungal agent with a novel mechanism of action with in vitro activity against Aspergillus fumigatus and other clinically important moulds. As antifungal combinations are of interest to extend the spectrum of coverage and improve antifungal activity, we investigated the pharmacodynamics of olorofim in combination with posaconazole. Methods Using galactomannan as a pharmacodynamic endpoint, olorofim and posaconazole were assessed alone and in combination in a neutropenic murine model of pulmonary aspergillosis using wild-type and triazole-resistant strains of A. fumigatus. Pharmacokinetic and pharmacodynamic data were fitted to a pharmacodynamic interaction model. Monte Carlo simulations of human-like regimens of both agents alone and in combination were performed to extrapolate to clinical settings. Results With the triazole-susceptible isolate, both monotherapy arms suppressed galactomannan, but suppression with the combination was less than expected from the monotherapy arms. With the triazole-resistant isolate, monotherapy produced galactomannan suppression with olorofim but not posaconazole; the combination arm produced less suppression than olorofim alone. The interaction model revealed antagonistic pharmacodynamic interaction parameter values. Extrapolations to human pharmacokinetics predicted that combination therapy would still have a net beneficial effect in A. fumigatus infections, albeit with reduced efficacy in infections with triazole-resistant isolates. Conclusions Posaconazole reduces the effect of olorofim in vivo. A combination of olorofim and a mould-active triazole is likely efficacious in wild-type infections but may be suboptimal in triazole resistance infections where there is minimal contribution of the mould-active triazole to antifungal activity and the triazole antagonises olorofim to produce a submaximal effect.