This study is based on a long-term field trial with spring barley monoculture that was established in 1970 on Gleyic Fluvisol in the Žabčice, Czech Republic. The aim was to clarify the long-term impact of straw management and mineral nitrogen (N) application on grain yields and soil aggregate stability (SAS), and to determine the mineralogical and geochemical properties crucial for soil aggregate stability changes. Variants of the experiment included a combination of incorporated and harvested straw with doses of 0, 30, 60, and 90 kg N ha⁻¹ in the form of ammonium sulphate (NH₄)₂SO₄. The incorporated straw variants had a higher average grain yield of 0.51 t ha⁻¹. The SAS values were in the range 54–64% and increased in all variants with N application compared to the 0N control. Ammonium sulphate fertilisation caused soil acidification, which was not reduced even by the incorporation of straw. SAS increased with decreasing pH value, although cation exchange capacity and exchangeable Ca²⁺ decreased, and the soil organic carbon content was similar in all variants. The relatively high content of Fe- and Al-(oxo)hydroxides extracted with ammonium oxalate (Fe_ox and Al_ox) in all samples caused an increase in SAS due to decreasing pH in the N fertilised variants compared to the control. SAS should be considered in relation to other soil properties when evaluating soil quality and fertility.