It is well known that the inhibition of cholinesterase (ChE) enzyme activity in metazoa is a specific biomarker of exposure to anticholinergic molecules such as neurotoxic drugs. These compounds destroy ChE activity by phosphorylating a specific serine residue within the ChE catalytic centre. However, the ChE activity is not peculiar to metazoa, as we have demonstrated its presence in protists, such as the ciliate Paramecium primaurelia and the sarcodine Dictyostelium discoideum, using different methodological approaches. The exploitation of protists as biotests for environmental risk assessment is widely accepted because they exhibit the fundamental features required for carrying out rapid analyses with a high degree of reproducibility. As eukaryotic organisms, protists behave like animals, responding directly to environmental stimuli. However, as single-cells, protists are more sensitive to environmental changes than the cells of higher organisms. Like micro-organisms generally, protists multiply through short cell-cycles and this makes it possible to detect the effects of xenobiotic compounds on large and genetically homogeneous cell populations and on their progeny as well. Both in P. primaurelia and D. discoideum, the inhibition of ChE activity has been found to be a valuable stress index of the presence of organophosphate and carbamate compounds, largely employed in rural areas for pest control. However, significant variations in ChE activity have been detected in single amoebae of D. discoideum exposed to heavy metals (CdCl₂ and HgCl₂), hydrocarbons (benzo-a-pirene), organochlorine drugs (dicofol) and extremely  low-frequency electromagnetic fields (200 mT,    50 Hz), thus suggesting that changes in ChE activity could represent a general biomarker of stress induced by environmental perturbations.