Insight is needed into how management influences soil biota when sustainable grassland systems are developed. A crop rotation of grass and maize can be sustainable in terms of efficient nutrient use. However, there is lack of information on the effect of such a crop rotation on soil biological quality. Earthworms, nematodes, bacteria and fungi were sampled over three years in a 36 years old experiment. Permanent arable land was compared with permanent grassland and with a ley-arable crop rotation. In the rotation, a period of three years of grassland (temporary grassland) was followed by a period of three years of arable land (temporary arable land) and vice versa. In the first year of arable cropping in the rotation, the number of earthworms was already low and not different from continuous cropping. In the three-year grass ley, the abundance of earthworms returned to the level of permanent grassland in the second year. However, the restoration of earthworm biomass took a minimum of three years. Furthermore, the anecic species did not recover the dominance they had in the permanent grassland. The numbers of herbivorous and microbivorous nematodes in the ley-crop rotation reached similar levels to those in the permanent treatments within one to two years. Although the same holds for the nematode genera composition, the Maturity Index and the proportion of omnivorous nematodes in the temporary treatments remained significantly lower than in their permanent counterparts. Differences in recovery were also found among microbial parameters. In the temporary treatments, bacterial growth rate and the capacity to degrade a suite of substrates recovered in the second year. However, the Community-Level Physiological Profiles in the permanent grassland remained different from the other treatments. Our results suggest that many functions of soil biota that are well established in permanent grassland, are restored in a ley-arable crop rotation. However, due to a reduction in certain species, specific functions of these soil biota could be reduced or lost. The ley-arable crop rotations were intermediate to permanent grassland and continuous arable land in terms of functioning of soil biota (e.g., N-mineralization). In terms of the functional aspects of the soil biota, permanent grassland might be preferable wherever possible. For maize cultivation, a ley-arable crop rotation is preferable to continuous arable land. However, a ley-arable crop rotation is only preferable to continuous arable cropping if it is not practised at the expense of permanent grassland at farm level.