Microbial methylation and demethylation are central to arsenic’s (As) biogeochemical cycling. Here, the transformations of monomethylarsonic acid (MMA(V)) (50 mgL−1) for 15 days in cells of As-methylating fungi, Fusarium oxysporum CZ-8F1, Penicillium janthinellum SM-12F4, and Trichoderma asperellum SM-12F1, were evaluated, and trace concentrations of As(III) and As(V) were observed in fungal cell extracts. Trace amounts of DMA(V) were also detected in MMA(V) and P. janthinellum SM-12F4 incubations. In situ X-ray absorption near edge structure (XANES) indicated that after exposure to MMA(V) (500mgL−1) for 15 days, 28.6–48.6% of accumulated As in fungal cells was DMA(V), followed by 18.4–30.3% from As(V), 0–28.1% from As(III), and 4.8–28.9% from MMA(V). The concurrent methylation and demethylation of As occurs in fungal cells. The findings of this study will develop our understandings of microorganisms that drive As speciation transformation.