Aspergillus fumigatus biofilm development results in enhanced pathogenicity and treatment resistance. Most contemporary antibiotics, however, are unable to eliminate biofilms. In recent years, with the application of new photosensitizers and the development of treatment, ALA-PDT (5-aminolevulinic acid photodynamic treatment) has achieved remarkable curative effect in the treatment of fungal infectious diseases; however, no research has been conducted on ALA-PDT against A. fumigatus. This study investigated the inhibitory effect of ALA-PDT at various 5-aminolevulinic acid concentrations and light doses on A. fumigatus planktonic and biofilms in vitro. We found that ALA-PDT may successfully inhibit the development of A. fumigatus biofilm and disintegrate mature biofilm. After ALA-PDT treatment, the adherence rate and vitality dramatically decreased, and the biofilm’s structure was severely compromised. Our findings show for the first time that ALA-PDT may be used to prevent the formation of A. fumigatus biofilm and disturb the structure of mature biofilm, and that it could be employed as a therapeutic therapy for A. fumigatus superficial infection.