Biological communities in the estuarine and shallow sediments have been continuously exposed to metal pollution and subsequent environmental stresses are of great concern due to persistent toxicity and non-biodegradable characteristics. While there have been many single species bioassay studies, few studies have addressed the community-level mesocosm studies in terms of benthic diatoms. For doing so, the reaction of marine benthic diatom communities to arsenic exposures has been investigated under controlled conditions of the mesocosms. Realistic concentrations (control, 5, 15, 45, and 135 ppm) of NaAsO2 powder have been spiked to the defaunated sediment followed by the inoculation of diatom biofilms then the mesocosms have been conducted for three weeks. In this study, biofilms had relatively As uptake capacity instead of total phosphorus along the arsenic exposure. Accordingly, the arsenic toxicity response of the diatom communities was observed in the aspect of community structure, and morphological abnormalities with categorized teratological type. The benthic diatom communities showed a shift in diatom assemblage composition by tolerance species (e.g., Cocconeopsis kantsinensis, Naviucla sp. 24, Naviucla sp. 18, Tryblionella hungarica), and sensitive species (e.g., Navicula gregaria and Austariella jamalinensis) over As exposure. Two-way PERMANOVA results indicated that changes in benthic diatom community composition were influenced significantly among time effects (df = 3, pseudo-F = 4.056, p < 0.0001), As treatment (df = 4, pseudo-F = 1.6459, p < 0.0012), and between time and As treatment (df = 12, pseudo-F = 1.539, p < 0.0001). The most dominant abundance was Cocconeiopsis kantsinensis, Navicula sp. 24, and Navicula gregaria, and diatom frustule deformities occurred frequently in Fragiliara crotonensis, Staurophora sp. 3, and Astartiella punctifera under the light microscope (LM) and scanning electron microscope (SEM). The diatom communities more often exhibited increased morphological deformation and various types upon As spiking. Regression analyses also showed positive correlation (r2 = 0.41) and strong statistical relationship (p < 0.003) between percent of deformed frustules and As concentration.