The antiozonant and research tool ethylene diurea (EDU) is widely studied as a phytoprotectant against the widespread pollutant ground-surface ozone. Although it has been extensively used, its potential toxicity in the absence of ozone is unknown and its mode of action is unclear. The purpose of this research was to toxicologically assess EDU and to further investigate its mode of action using Lemna minor L. as a model organism. Application of EDU concentrations greater than 593 mg L−1 (practically 600 mg L−1) resulted in adverse inhibition of colony growth. As no-observed-toxic-effects concentration (NOEL) we recommend a concentration of 296 mg L−1 (practically 300 mg L−1). A hormetic response was detected, i.e. stimulatory effects of low EDU concentrations, which may indicate overcompensation in response to disruption in homeostasis. Growth inhibition and suppressed biomass were associated with impacted chlorophyll a fluorescence (ΦPSII, qP and ETR). Furthermore, EDU increased mesophyll thickness, as indicated by frond succulence index. Applications of concentrations ≥593 mg L−1 to uncontrolled environments should be avoided due to potential toxicity to sensitive organisms and the environment.