In environmental and soil sciences, bioavailability represents the amount of an element or compound that is accessible to an organism for uptake or adsorption across its cellular membrane. In agricultural applications, almost exclusively plant roots and soil organisms uptake contaminants that are dissolved in the soil water. On this basis, a study was conducted to determine the relationship between diuron bioavailability to plants or microorganisms of aged herbicide residues in 10 selected soil types, and their extraction from these soils using three different solutions: n-butanol, hydroxypropyl-beta-cyclodextrin (HPBCD) and CaCl2. The aim of the mineralisation bioassays was to assess the diuron bioavailable fraction mineralised by a diuron soil microbial degrader consortium. The bioassays using plants aimed to assess the diuron biotoxicity employing a selected crop (Hordeum vulgare). Toxicity was determined as the level at which plant growth was inhibited. Aging of diuron contaminated soil during 100 days drastically decreased the percentage of diuron recovered by the selected extractants in all the tested soils. Results showed statistical correlations between non-exhaustive chemical diuron extraction and biological measurements when the extractant was n-butanol, showing to be the most reliable chemical technique capable of predicting both, diuron bioavailability for microorganisms and for plants. HPBCD was also capable of detecting diuron bioavailability only for microorganism, although with worse statistical data, while the third extractant, CaCl2 aqueous solution, showed to be the least reliable for both assays. The results presented demonstrated that a non-exhaustive chemical extraction using n-butanol as extractant may serve as the basis for a simple and rapid procedure to assess diuron bioavailability in contaminated soils with different characteristics.