Tillage and N fertilization practices contribute to the balance between soil C inputs and outputs. Thus, the impacts of both practices and their interactions on soil organic C (SOC) dynamics must be studied. The main objective of this study was to determine long-term effects of tillage and N fertilization on soil biochemical properties in a long-term experiment established in 1996 on a dryland Typic Xerofluvent soil cropped with barley (Hordeum vulgare L.) in NE Spain. The response of SOC concentration, soil microbial biomass carbon (SMBC) and soil enzyme activities (DHA, dehydrogenase, and PRA, protease) to different tillage (no-tillage, NT; reduced tillage, RT; and conventional tillage, CT) and N treatments (zero, 0kg Nha −1 ; medium, 60kg Nha −1 ; and high, 120kg Nha −1 ) were measured in 2008 at four soil depths (i.e., 0–5, 5–10, 10–25 and 25–50cm). All the soil biochemical properties studied showed significant differences for tillage, depth and the interaction between tillage and soil depth. However, N fertilization rates only affected the SMBC content, which was greater under 120kg Nha −1 than under 0kg Nha −1 in the 10–25cm soil layer. In the soil surface layer (0–5cm), SOC, SMBC and DHA levels in CT were about 50% of the levels in the NT plots. However, in the 10–25cm soil layer, a greater SOC concentration in CT compared with NT and RT was also accompanied by SMBC and DHA values 30% higher in CT. Below 25cm soil depth, similar values of soil biochemical properties were found among tillage systems. There was a significant correlation among almost all the parameters studied, with the greatest correlations between SOC and SMBC and between SOC and DHA. In semiarid Mediterranean conditions, after 12 years of experiment, tillage impacted soil biochemical properties in a greater extent compared with N fertilization even though this effect was only limited to the upper soil layers.