As widely used in many kinds of products, silver nanoparticles (AgNPs) are inevitably released into the aquatic environment. Constructed wetlands (CWs) are essential sinks of AgNPs, while little is known about the responses of microorganisms (biofilm on the substrate surface) and hydrophyte functions to AgNPs accumulation in CWs. Given that, we conducted a 60-day experiment exposing to 1 and 50 mg L−1 of AgNPs in CWs. Results revealed that AgNPs accumulation obviously decreased the relative abundance of genes involved in biofilm formation and microbial nitrogen metabolism, whereas increased those in the glycolysis process; AgNPs also decreased key enzyme activities corresponding to nutrient removal. In addition, AgNPs accumulation decreased the hydrophyte activity, causing a decline in the uptake ability of C (6%–44%), N (17%–54%), and P (14%–56%) elements. Finally, due to the decline in the whole biofilm and hydrophyte functions under AgNPs exposure, the treatment performance of CWs significantly decreased by 40%–56% of total nitrogen and 44%–60% of ammonia nitrogen, respectively. Overall, our results first revealed biofilm and hydrophyte's functional response to AgNPs exposure. We systematically analyzed the changes of vital functional modules of microorganisms based on the metagenomic analysis, as well as the functional emergency response of aboveground and underground tissues of hydrophyte in CWs under AgNPs stress.