In this work, three pesticides of different physicochemical properties, namely, glyphosate (herbicide), imidacloprid (insecticide) and imazalil (fungicide), were selected to assess their cytotoxicity against distinct cell models (Caco-2, HepG2, A431, HaCaT, SK-MEL-5 and RAW 264.7 cells) to mimic gastrointestinal and skin exposure with potential systemic effect. Cells were subjected to different concentrations of selected pesticides for 24 h or 48 h. Cell viability was assessed by Alamar Blue assay, morphological changes by bright-field microscopy and the IC50 values were calculated. Cytotoxic profiles were analysed using the physico-chemical parameters of the pesticides, namely: molecular weight, water solubility, the partition coefficient in the n-octanol/water (Log Pow) system, the topological polar surface area (TPSA), and number of hydrogen-bonds (donor/acceptor) and rotatable bonds. Results showed that glyphosate did not reduce cell viability (up to 1 mM), imidacloprid induced moderate toxicity (IC50 > 1 mM for Caco-2 cells while IC50 = 305.9 ± 22.4 μM for RAW 264.7 cells) and imazalil was highly cytotoxic (IC50 > 253.5 ± 3.37 for Caco-2 cells while IC50 = 31.3 ± 2.7 μM for RAW 264.7 cells) after 24 h exposure. Toxicity was time-dependent as IC50 values at 48 h exposure were lower, and decrease in cell viability was accompanied by changes in cell morphology. Pesticides toxicity was found to be directly proportional with their Log Pow, indicating that the affinity to a lipophilic environment such as the cell membranes governs their toxicity. Toxicity is inverse to pesticides TPSA, but lower TPSA favours membrane permeation. The lower toxicity against Caco-2 cells was attributed to the physiology and metabolism of cell barriers equipped with various ABC transporters. In conclusion, physicochemical factors such as Log Pow, TPSA and H-bond are likely to be directly correlated with pesticide-induced toxicity, thus being key factors to potentially predict the toxicity of other compounds.