Aspergillus fumigatus biofilms still present a challenge for effective treatment in clinical settings. While mild heat stress has been introduced as a treatment for infectious diseases, the effectiveness of mild heat stress on A. fumigatus biofilm formation and antifungal susceptibility is still unknown. In the present study, confocal laser scanning microscopy (CLSM) was used to image and quantify Aspergillus fumigatus biofilm formation under three different regimens of continuous mild heat stress: at 37, 39, and 41°C. Furthermore, fungal growth has been investigated under the above conditions in combination with antifungal drugs (amphotericin B [AMB], micafungin [MCF], and voriconazole [VOC]) at early and late stages. CLSM analysis showed that higher temperatures induce earlier germination and greater hyphal elongation but poorer polar growth and reduced biofilm thickness. In the early stage of biofilm formation, the combination of treatment at 39 or 41°C with MCF or VOC produced no visible difference in biomass formation from similar treatments at 37°C with the same drug. Interestingly, AMB treatment at 37°C inhibited early stage biofilm formation to a much greater extent than at 39 and 41°C. At the late stage of biofilm formation, the mild heat treatments at 39 and 41°C with AMB, MCF, and VOC inhibited biomass formation compared to that at 37°C. The present data show that mild heat stress has a negative regulatory effect on biofilm formation in vitro, and antifungal drug improvement with mild heat treatment at late-stage biofilm formation provides useful indications of possible effective strategies for clinical management of aspergillosis.