The impact of diazinon spraying in an agricultural tropical soil through the evaluation of both the habitat and retention functions of the soil system was never reported. To fill this gap, five times the recommended dose of a commercial diazinon formulation was sprayed in an agricultural area of Costa Rica, and dilution gradients of the sprayed soil were prepared in the laboratory. Avoidance and reproduction tests with soil organisms (Eisenia andrei, Enchytraeus crypticus and Folsomia candida) to evaluate losses in terrestrial habitat function, and growth and reproduction tests with aquatic organisms (Chlorella vulgaris and Daphnia magna, respectively) to evaluate the retention function of soil were performed. Results demonstrated that regarding habitat function, F. candida reproduction was the most sensitive endpoint (EC(50) = 0.288 mg a.i./kg), followed by avoidance behaviour of E. andrei (EC(20) = 1.75 mg a.i./kg). F. candida avoidance and the reproduction of E. andrei and E. crypticus were not affected by diazinon. The toxicity tests with aquatic organisms showed that the soil retention function was insufficient to prevent effects of diazinon either on microalgae growth (EC(50) ≤ 0.742 mg/L and EC(20) ≤ 0.223 mg/L) and on the reproduction of the cladoceran (EC(50) ≤ 0.00771 mg/L and EC(20) ≤ 0.00646 mg/L). Results suggested that diazinon exerted toxic effects even at the dilution corresponding to the recommended dose, fact which makes its misuse an issue of environmental concern. Furthermore, the present study highlighted the importance and complementary nature of the assessment of both habitat and retention functions to an ecological risk assessment in tropical systems.