Conservation agriculture based on no-tillage (NT) and crop rotation allows to enhance soil health. Based on data collected from long-term trials in a semi-arid region of Tunisia, results showed that NT increased significantly soil organic carbon stock (SOCS), soil microbial biomass carbon (SMBC), arbuscular mycorrhizal fungal (AMF) root colonization, and soil microbial respiration (CO₂) at 0–20 cm topsoil layer compared to conventional tillage (CT). Moreover, triennial rotation (TRI), based on annual succession of Faba bean-Durum wheat-Barley, and biennial rotation (BI), based on annual succession of Faba bean-Durum wheat, increased significatively SMBC, AMF, and CO₂. Likewise, a significant benefit of the two-way interactions Tillage × Rotation was observed. Furthermore, NT combined with TRI recorded the highest SOCS (2181 g C m⁻²), SMBC (515 mg C kg⁻¹ soil), AMF (14%), and CO₂ which is an indicator of soil microbial respiration (1071 mg CO₂ kg⁻¹ soil). The current results highlight the benefit adoption of minimum or (NT)combined with crop diversification on soil health.