Abstract Background and objectives The emergence of azole resistance among Aspergillus fumigatus isolates has driven the development of novel antifungal compounds, which may potentially be used in combination with existing antifungal drugs. In this study, we evaluated the in vitro interaction between olorofim, a novel orotomide antifungal, and voriconazole, a triazole agent, against A. fumigatus using two complementary approaches. Methods A liquid microdilution checkerboard assay was performed in duplicate on 32 clinical isolates, including 27 voriconazole-susceptible and 5 voriconazole-resistant isolates, to determine the MICs of both drugs alone and in combination. Interactions were quantified using the FIC index and through response surface modelling based on the Bliss independence model. Furthermore, an agar-based method was conducted on a subset of 11 isolates. Media were supplemented with either voriconazole or olorofim, on which discs containing the reciprocal drug were applied. Additionally, interaction was assessed by using Etest® strips of voriconazole on olorofim-containing agar. Results Antagonism was observed for 56% of isolates based on the checkerboard technique and for 75% of the isolates based on response surface analysis. Antagonistic interaction was observed for all the voriconazole-resistant isolates. Across all tested isolates and agar configurations, antagonistic interactions were consistently observed, including reduced inhibition zones around olorofim discs in the presence of voriconazole and paradoxical fungal growth surrounding voriconazole discs or strips on olorofim-containing plates. Conclusions These findings demonstrate the in vitro antagonism between olorofim and voriconazole and underscore the need for further investigation before considering their combined clinical use.