Abstract It was shown that the use of biochar provides many benefits to agriculture by improving the whole complex of soil properties, including soil structure. However, the diverse range of biochar effects depends on its physicochemical properties, its application rates, soil initial properties etc. The impacts of biochar, mainly its reapplication to soils and its interaction with nitrogen in relation to water-stable aggregates (WSA) did not receive much attention to date. The aims of the study were: (1) to evaluate the effect of initial application (in spring 2014) and reapplication (in spring 2018) of different biochar rates (B0, B10 and B20 t ha−1) as well as application of biochar with N-fertilizer (40 to 240 kg N ha−1 depending on the requirement of the cultivated crop) on the content of WSA as one of the most important indicators of soil structure quality, (2) to assess the interrelationships between the contents of soil organic matter (SOM) and WSA. The study was conducted in 2017–2019 as part of the field experiment with biochar on Haplic Luvisol at the experimental station of SUA in Nitra, Slovakia. Results showed that initial application as well as reapplication of biochar improved soil structure. The most favorable changes in soil structure were found in N0B20B treatment (with biochar reapplication) at which a significantly higher content of water-stable macro-aggregates (WSAma) (+15%) as well as content of WSAma size fractions of > 5 mm, 5–3 mm, 3–2 mm and 2–1 mm (+72%, +65%, +57% and +64%, respectively) was observed compared to the control. An increase in SOM content, due to both, initial biochar application and its reapplication, significantly supported the stability of soil aggregates, while organic matter including humic substances composition did not.