Phenol is a ubiquitous environmental pollutant and a widely used reference toxicant for many bioassays. However, little information is available regarding the toxic effects of phenol on aquatic macrophytes. Seventy-two hour bioassays, with different end-points, were carried out to assess phenol toxicity in Lemna paucicostata. A concentration-dependent decline in frond multiplication and colony disintegration was observed, with 11.38 and 22.76 μM phenol resulting in browning of fronds and colony disintegration, respectively. Growth of fronds, as measured by changes in surface area, was significantly inhibited with EC 50 value of 2.70 μM. When pulse amplitude modulated chlorophyll a (Chl a) fluorescence imaging (i-PAM) was employed, the maximum quantum yield of PS II ( F v/ F m) significantly declined with increasing phenol concentrations with resultant EC 50 of 1.91 μM and coefficients of variation (CVs) generated for the EC 50 values of less than 4.7%. A gradual increase in fluorescence emissions from chlorophylls a and b and pheophytin up to a concentration of 2.85 μM was found but declined markedly at higher concentrations. The significant correlation between the F v/ F m and surface growth rate data implies that the former is an appropriate biomarker of whole plant toxicity. Using imaging Chl a fluorescence on L. paucicostata provides a rapid, sensitive and reliable method for assessing the toxic risks posed by phenol to aquatic ecosystems and has practical applications for municipal and industrial waste water management.