Biofilm production by microorganisms is critical for their pathogenicity. Serum promotes biofilm production by Aspergillus fumigatus; however, its effects on other Aspergillus spp. have not been reported. We analyzed biofilm formation by five Aspergillus spp., i.e., A. fumigatus, A. flavus, A. nidulans, A. niger, and A. terreus, and examined the effects of serum/serum proteins such as fetal bovine serum (FBS), fetuin A, and bovine serum albumin (BSA) on hyphal growth, hyphal branching, and extracellular matrix (ECM) formation. The antifungal susceptibility of A. fumigatus isolates that formed biofilms was also examined. All serum/serum proteins promoted the growth of all these fungal species; growth promotion was most evident with FBS, followed by fetuin A and BSA. This effect was most evident in case of A. fumigatus and least evident in case of A. terreus. Electron microscopy showed thick ECM layers surrounding fungal cell walls after culture with FBS, particularly in A. fumigatus. An increase in hyphal branching caused by fetuin A was the highest in case of A. fumigatus and A. nidulans. Biofilm-forming A. fumigatus showed resistance to most antifungal agents, although a synergism of micafungin and amphotericin B was suggested. Our results indicate that serum promotes biofilm formation, including thick ECM, by many Aspergillus spp., particularly A. fumigatus, and that this may be closely related to its virulence.