Background and Aims: Organic farmers are showing increasing interest in using conservation tillage to improve the biological activities of soils. Here, we assessed whether conservation tillage in organic farming improves earthworm populations, root growth and soil physical quality in a sandy loam after 16 years of experiment. Methods: We compared the effect of a tillage gradient, with of two non-ploughed treatments (superficial tillage [ST] at 15 cm; very superficial tillage [VST] at 5–7 cm) and two ploughed treatments (moldboard ploughing [MP] at 30 cm; shallow moldboard ploughing [SMP] at 20 cm). Soil clod types, penetration resistance, abundance and activity of earthworms, root traits and biomass were assessed. Results: VST decreased soil compaction in topsoil (0 to 10 cm) compared to ploughed treatments (MP and SMP), but led to more compacted soil at 15 to 30 cm. Earthworm biomass (especially anecic) was higher under VST compared to MP and SMP and their galleries were better connected to the soil surface. However, there was no significant difference in the total volume of pores or diameter of galleries between 0 and 30 cm. Soil compaction in the non-ploughed treatments affected root traits, especially under VST, with lower specific root length, higher root diameter, and lower root tip elongation compared to MP and SMP. Conclusion: Biological activity did not compensate for the compaction of a sandy soil after 16 years without ploughing in organic farming. A more integrated approach (i.e. considering all 5 soil health principles) is needed to sustain soil health and functions, and meet current expectations about “ecological intensification”.