Hydrilla verticillata (waterthyme) has been successfully used for phytoremediation in arsenic (As) contaminated water. To evaluate the effects of environmental factors on phytoremediation, this study conducted a series of orthogonal design experiments to determine optimal conditions, including phosphorus (P), nitrogen (N), and arsenate (As(V)) concentrations and initial pH levels, for As accumulation and biotransformation using this aquatic plant species, while also analyzing As species transformation in culture media after 96-hr exposure. Analysis of variance and the signal-to-noise ratio were used to identify both the effects of these environmental factors and their optimal conditions for this purpose. Results indicated that both N and P significantly impacted accumulation, and N was essential in As species transformation. High N and intermediate P levels were critical to As accumulation and biotransformation by H. verticillata, while high N and low P levels were beneficial to As species transformation in culture media. The highest total arsenic accumulation was (197.2 ± 17.4) μg/g dry weight when As(V) was at level 3 (375 μg/L), N at level 2 (4 mg/L), P at level 1 (0.02 mg/L), and pH at level 2 (7). Although H. verticillata is highly efficient in removing As(V) from aquatic environments, its use could be potentially harmful to both humans and the natural environment due to its release of highly toxic arsenite. For cost-effective and ecofriendly phytoremediation of As-contaminated water, both N and P are helpful in regulating As accumulation and transformation in plants.