Soil and crop management influence soil organic carbon (SOC), chemical composition, and overall soil quality. In a Mediterranean region, a study initiated in 1994 examined the long-term effects of conventional tillage (CT) versus no-tillage (NT) practices. Initially focusing on continuous durum wheat cultivation until 2009, the experiment later introduced a 2-year rotation of durum wheat and Vicia faba L. cover crops in half of the CT and NT fields. SOC was monitored from 2008 to 2018, while microbial biomass (as dsDNA), soluble nitrogen (N), and enzyme activities (EAs) were monitored from 2011 to 2014 to evaluate the rotation’s impact. Between 2009 and 2018, CT yields were on average 15% higher than NT, especially during high rainfall years. NT significantly increased SOC content in the 0–30 cm soil layer, along with higher levels of soluble N, dsDNA, and EAs at 0–10 cm depth. NT led to a 23% and 10% increase in SOC stock and SOC stock per equivalent soil mass compared to CT. EAs increased by over 50% under NT, indicating enhanced biological activity. The SOC increase due to NT was limited to the top 10 cm, with a decrease at deeper depths (up to 50 cm). Introducing cover crops over 4 years did not yield significant impacts, suggesting the need for a longer period to observe noticeable effects. Overall, adopting NT practices resulted in higher SOC concentration, enhanced soil biological activity, and improved biogeochemical cycles, emphasizing the positive impact of NT on soil health and sustainability.