The interaction between polyenes and azoles is not well understood. We therefore explored the in vitro combination of amphotericin B with itraconazole against 14 clinical Aspergillus fumigatus isolates (9 itraconazole susceptible and 5 itraconazole resistant) with a colorimetric broth microdilution checkerboard technique using two drug interaction models able to explore complicated patterns of interactions: the response surface analysis of Bliss independence and the isobolographic analysis of Loewe additivity zero interaction theories. Synergy was found at combinations with low concentrations of amphotericin B (<0.125 mg/L), whereas antagonism was found at combinations with higher concentrations of amphotericin B. For itraconazole-resistant isolates, synergistic interactions were observed at high concentrations of itraconazole (>0.5 mg/L). Synergy was more frequently observed for the itraconazole-resistant isolates than for the itraconazole-susceptible isolates.