Ultrasonication is widely used in soil organic matter (SOM) fractionation studies to break up soil aggregates to disperse the soil into its primary particles. An increasing number of studies also aim to incubate SOM fractions obtained by physical soil fractionation to study the bioavailability of different soil organic C (SOC) pools. To evaluate possible influences of ultrasonic soil disruption on short-term SOM bioavailability as well as the content and composition of water-extractable organic matter (WEOM) and salt-extractable (10 mM K₂SO₄) organic matter (SEOM), we conducted a laboratory incubation experiment with aggregated soil material from an Ap horizon of a cropland soil. Bulk soil and subsamples in which aggregates had been disrupted by ultrasonication were incubated in triplicate for 18 d at 20°C. During the incubation, CO₂ production was measured continuously. At the beginning and at the end of the experiment, the chemical composition of SEOM was analyzed for the different experimental variants by ultraviolet (UV)-absorbance and solid state ¹³C cross polarization magic angle spinning nuclear magnetic resonance (¹³C-CPMAS NMR) spectroscopy. Additionally, we studied the effects of WEOM removal on heterotrophic soil respiration to assess artifacts by unavoidable WEOM removal during soil wet sieving, which is an important step in soil fractionation. After repeated leaching of unsonicated and sonicated soil samples, 0.5 to 1.0% of the total organic C (OC) pool was associated with the WEOM fraction. Ultrasonic disruption of soil aggregates resulted in increased amounts and changed composition of SEOM as well as in a 27% increase in CO₂ production compared with intact soils. Incubation of ultrasonicated samples resulted in decreased O/N alkyl C and increased aromatic C contents in SEOM, indicating mineralization of potentially mobile organic matter (OM). Ultrasonic treatment during soil fractionation leads to enhanced dissolved OM (DOM) leaching and increased SOM bioavailability, which may bias results from incubation experiments on separated SOM fractions.