Sterilized soil is often used, for example in degradation studies, sorption experiments, microbiological tests and plant test systems, to distinguish between microbial processes and abiotic reactions. The most commonly used technique for sterilization is autoclaving of the soil. Another technique is irradiation with high-level gamma radiation (γ-radiation). One major drawback of sterilization procedures is the possible alteration of the structure of soil components, for example the organic matter. A change in the chemical structure of the soil organic matter can cause different reactions in the above-mentioned experiments and hence interfere with the aim of clearly distinguishing between biotic and abiotic processes. Two soils (Gleyic Cambisol and Orthic Luvisol) were sterilized by two γ-irradiation procedures (4 kGy hour−1 for 9 hours and 1.3 kGy hour−1 for 27 hours) and repeated autoclaving at 121°C. Gentle physical aggregate fractionation of the sterilized soils revealed a decrease in the aggregation of the soil, which was reflected in an increase of the clay fraction. Subsequent analysis of the aqueous phase revealed much more dissolved organic matter (DOM) in the γ-sterilized and autoclaved soils than in the untreated soils. Ultraviolet (UV) and fluorescence spectra of the DOM showed a decrease in the aromaticity and polycondensation of the dissolved organic carbon (DOC). 13C cross-polarization/magic-angle spinning nuclear magnetic resonance (13C-CP/MAS NMR) spectra of the unfractionated soils and their respective soil fractions before and after sterilization showed that the most important change occurred in the carbohydrate and N-alkyl region, the main components of microorganisms. In general, the impact of the sterilization method was stronger for autoclaving. The γ-sterilized soils and fractions displayed both fewer and smaller changes in the soil organic matter.