Plants and microorganisms are the main biotic compartments for phytoaccumulation and metabolic transformation of organic contaminants in constructed wetlands (CWs). However, how they cope with special pollutants during the treatment process has not been well characterized. In this study, responses of Iris pseudoacorus (I. pseudoacorus) and microbial communities were intensively investigated in pilot-scale CWs treating wastewater-borne chlorpyrifos, an organic phosphorus pesticide. Chlorpyrifos was associated with inhibited plant growth, decreased photosynthetic activity, and a significant increase in oxidative products. Superoxide dismutase (SOD) and ascorbate peroxidase (APX) activities were affected by chlorpyrifos, whereas catalase (CAT) activity was almost unaffected. Although chlorpyrifos stimulated the antioxidant system, there was little indication of oxidative damage in I. pseudoacorus. Urease, β-glucosidase, and phosphatase activities in substrate were elevated by 73.73%, 17.20%, and 16.23%, respectively, which may indicate enhancement of nitrogen, carbon, and phosphorus cycling. Fatty acid methyl ester (FAME) profiles showed that aerobic prokaryotes, which are likely the functional group responsible for chlorpyrifos degradation, increased from 16.37% to 31.32% after chlorpyrifos addition. Biomarkers for fungal/bact and trans/cis remained unchanged after the chlorpyrifos addition, suggesting that chlorpyrifos did not negatively influence the substrate microbial communities in CWs.