Soils under no-till (NT) in Brazil have been aimlessly chiseled mainly due to compaction symptoms. The impacts on soil organic carbon (SOC) of these operations as well as the effects of more sustainable alternatives are still highly unknown. We hypothesized that the use of plants with deep root systems in the crop rotation could be a suitable alternative to mechanical chiseling for no-till fields. Thus, our objective in this study was to evaluate changes in SOC stocks, root development, and crop yield over time caused by mechanical and biological chiseling in a long-term no-till system (i.e., 18 years). The experiment was laid out as randomized blocks with four replicates and the treatments were as follows: 1) No-till system without any mechanical disturbance, cultivated with black oats + vetch in winter, here called “No-till cover crop (NT-CC)”. 2) No-till system with one mechanical disturbance through chiseling, also cultivated with black oats + vetch in winter, here called “No-till Mechanical Chiseling (NT-MC)”. 3) No-till without any mechanical disturbance cultivated with black oats + forage radish (Raphanus Sativus L.) instead of vetch, here called “No-till Biological Chiseling”. Compared to NT-CC, the use of NT-MC depleted the SOC stocks by -0.41 Mg ha⁻¹ year⁻¹. NT-MC also highly depleted proxies for microbial-related C pools such as hot-water and permanganate extractable C. It demonstrates the deleterious effects of the mechanical chiseling operations on the surface depths (0−30 cm depth) of our long-term no-till system and raises concerns about the use of these operations in highly weathered areas. On the other hand, the NT-BC promoted significantly higher sequestration rates of 1.29 Mg ha⁻¹ year⁻¹ (LSD test, p < 0.05). The NT-BC has also promoted significantly higher sequestration rates of proxies of microbial-related organic matter, and soybean and wheat root development (length and mass) than the other managements. For cumulative crop yield and biomass input, both NT-BC and NT-CC promoted significantly higher values than NT-MC during the four years of assessment. The root development of wheat and soybean significantly contributed to the increase of SOC stocks, observed by positive correlations (p < 0.01). The results demonstrated that the use of radish as an intercrop for alleviating soil compaction was a good alternative to replace mechanical chiseling in compacted NT fields due to its superior performance in increasing SOC stocks, promoting higher root development, and crop yield.