Background Optimizing antifungal therapy is important to improve outcomes in severely immunocompromised patients. Objectives We analysed the in vitro interaction between pulmonary surfactant and antifungal agents used for management of invasive pulmonary aspergillosis. Methods Amphotericin B formulations, mould-active triazoles and echinocandins were tested in vitro against 24 clinical isolates of different Aspergillus spp. with and without the addition of a commercial porcine surfactant (Curosurf®; Poractant alfa, Nycomed, Austria). The data are presented as MIC or minimum effective concentration (MEC) ranges, as MIC or MEC values that inhibited 90% of the isolates (MIC90 or MEC90) and as geometric mean (GM) MIC or MEC values. Results For amphotericin B products, addition of surfactant to a final concentration of 10% led to a statistically significant reduction of the GM MIC for all Aspergillus isolates tested after 24 h (0.765 versus 0.552 mg/L; P < 0.05). For the mould-active triazoles, addition of 10% surfactant resulted in a significantly higher GM MIC at 48 h (0.625 versus 0.898 mg/L; P < 0.05). For the echinocandins, the addition of 10% surfactant led to a significantly higher GM MEC after both 24 h (0.409 versus 0.6532 mg/L; P < 0.01) and 48 h (0.527 versus 0.9378 mg/L; P < 0.01). There were no meaningful differences between individual members of the three existing classes of antifungal agents or between the different Aspergillus spp. tested. Conclusions Using EUCAST methodology, addition of porcine surfactant up to a concentration of 10% had a minor, and presumably non-relevant, impact on the in vitro activity of antifungal agents used in prophylaxis and treatment of invasive pulmonary aspergillosis.