Optimization of fertilizer and tillage management in agroecosystems can be an effective strategy to reduce greenhouse gas (GHG) emission and improve carbon sequestration. Using a system boundary that started at winter wheat seeding and ended at summer maize harvest, a field experiment was conducted in Shandong Province (North China) to determine the effect of microbial fertilization and tillage on the carbon footprint (CF). The climate-based ecosystem services were evaluated in wheat-maize cropping systems. Also the optimal microbial fertilization and tillage with the lowest CF, highest climate-based ecosystem services and highest crop yield were determined. The results showed that compared with microbial organic fertilization, the use of microbial decomposing agents increased soil organic carbon (SOC) sequestration (2582.1–2909.9 kg CO2-eq ha−1 yr−1) and reduced CF (0.11–0.12 kg CO2-eq kg−1 grain). Chemical fertilization was the largest contributor to total CF (27.5–56.7%), which decreased with decreasing chemical fertilization. Climate-based ecosystem services of microbial fertilization with rotary tillage (1689.9–1795.6 US$ ha−1 yr−1) were significantly higher than those of chemical fertilization. In the limited system boundary of wheat-maize cropping, microbial decomposing agent MA (of 30 L ha−1) with rotary tillage had the highest grain production, GHG emission mitigation and climate-based ecosystem services enhancement.