Background Wastewater treatment plant outlets are a major source of microplastics, with more than 90% retained in sewage sludge. No standardised method for the extraction, quantification, and characterisation of microplastics in sewage sludge or soil exists, and direct comparison of studies is often impossible. Our aim was to validate oil extraction efficiency with and without pre-treatment with Fenton’s reagent of selected microplastics in various types of environmental samples (sewage sludge and organic-rich substrates). Results Oxidation with Fenton’s reagent removed up to 90% of organic material, which improves the recovery rate and made quantification and characterisation easier and more reliable, regardless of type, shape, size, or density of the selected microplastic particles used in this study. Pre-treatment, as a pre-step of the oil extraction method, was shown to be important in reducing organic matter in all environmental samples, including sewage sludge and organic-rich substrates. It also improved the reliability of the selected method, shortened its duration, and, by reducing organic matter, made extracted microplastics more visible. The recovery rate was better for particles 1–5 mm and lower for particles 0.1 < 1 mm. Conclusions By achieving up to a 100% recovery rate for certain types of microplastics (polypropylene and polystyrene), the selected method proved to be a promising extraction method. It was also shown to be efficient in the organic-rich substrates, for which the characterisation of microplastic particles was done by Fourier transform infrared spectroscopy. The most commonly detected types of microplastics in organic-rich substrates were polyethylene, polypropylene, polystyrene and polyester.