Chitin, from the fungal source (Ganoderma lucidum spore powders, GLSP), was converted into chitosan via dual-frequency ultrasound irradiation which produced enhanced results compared with single-frequency processing. The differences between the effects of dual-frequency ultrasound irradiation (at 15 kHz and 20 kHz) with two superposition modes, in orthogonal and parallel orientations, were studied. SEM images confirmed morphology change in the presence of the dual-frequency sources. The enhancement of the degree of deacetylation (DD), dynamic viscosity ([η]) and molecular weight (Mv¯) of the resultant chitosan were also improved by the orthogonal configuration for dual-frequency ultrasound irradiation. In addition, the FTIR, TGA, XRD and 13 C NMR results show the differences in chemical groups, thermal stability and crystalline using two different ultrasound conditions in detail. The resulting biocompatible sample improved the proliferation of L929 cells, while antibacterial activity was also observed using E. coli and S. aureus. This presents a promising new use of a fungal material for biomedical applications.