HadA monooxygenase is involved in the initial step of the biodegradation pathway of toxic nitrophenols and halogenated phenols. HadA catalyzes the O2‐dependent denitration of nitrophenols and dehalogenation of halogenated phenols via the hydroquinone pathway. Based on bioinformatics and structural analysis, Arg208 of HadA is located at the proper position for substrate stabilization. This arginine is conserved among hydroquinone pathway‐specific enzymes for toxicant detoxification. In this study, the function of Arg208 in HadA was determined by a single‐point mutation creating HadAArg208 variants. 4‐Nitrophenol was mineralized by HadAArg208 variants that contain side chains as a positive charge and hydrogen‐bond donor, whereas 4‐chlorophenol strictly required a positively charged environment for detoxification. Transient kinetic results indicated that the biodetoxification ability of HadAArg208 variants was diminished due to the slowing down of denitration/dehalogenation. The substrate‐binding mode and affinity energy were evaluated by molecular docking. The findings were consistent with the experimental results indicating that arginine is the most fit for both 4‐nitrophenol and 4‐chlorophenol binding, whereas the active mutants provide a weaker interaction correlated with their denitration/dehalogenation activities. Altogether, Arg208 plays a role in providing proper chemical interactions to the substrate for binding at an appropriate orientation in the active site of hydroquinone pathway‐specific enzymes. In addition, it is proposed to stabilize nitro groups and halide ions that are released in denitration/dehalogenation reactions. This conserved arginine might be the essential feature for related biocatalysts, which could be fundamental knowledge regarding this enzyme family.