Deep earthwork activities carried out before vineyard plantation can severely affect soil profile properties. As a result, soil features in the root environment are often much more similar to those of the underlying substratum than those of the original profile. The time needed to recover the original soil functions is ecologically relevant and may strongly affect vine phenology and grape yield, particularly under organic viticulture. The general aim of this work was to investigate soil resilience after vineyard pre-planting earthworks. In particular, an old and a new vineyard, established on the same soil type, were compared over a 5-year period for soil chemical, physical, micro- and mesobiological properties. The investigated vineyards (Vitis vinifera L., cv. Sangiovese) were located in the Chianti Classico district (central Italy), on stony and calcareous soils, and were not irrigated. The older vineyard was planted in 2000, after slope reshaping by bulldozing and back-hoe ploughing down to about 0.8–1.0 m. The new vineyard was planted in 2011, after equivalent earthwork practices carried out in the summer of 2009. Both vineyards were organically managed, and they were fertilized with compost only every autumn (1000 kg ha−1 per year). The new vineyard was cultivated by periodic tillage, while the old vineyard was managed with alternating grass-covered and tilled inter-rows. Soil samples were collected at 0–15 cm depth from fixed locations in each vineyard every spring from 2010 to 2014. The old vineyard was sampled in both tilled and grass-covered inter-rows. According to the results from physical and chemical analyses, the new vineyard, during the whole 2010–2014 period, showed lower total organic carbon, total nitrogen, carbon to nitrogen ratio and electrical conductivity, along with higher silt and total CaCO3 contents than the old vineyard, suggesting still-evolving equilibrium conditions. The microarthropod analysis showed significantly different abundances and community structures, in relation to both vineyard and time. Rainfall appeared to have an enhancing effect on microarthropod abundance, but only in the old vineyard, where the biota was more structured than in the new one. The euedaphic forms, well adapted to soil life, were always rare. Microbiological analysis revealed a different structure of eubacterial communities between the old and the new vineyard in the whole period. However, the DGGE similarity values of these communities increased by about 2.5% per year, suggesting that at least 3 years more are needed to compare intra- and inter-specific diversity of the two vineyards. In conclusion, the consequences of deep earthworks on soil chemical, micro- and mesobiological properties were still evident 4 years after planting, indicating that more time is necessary for the recovery of soil functions, probably longer than the time needed to reach a state of economically viable grape production.