AbstractMacro-organisms contribute significantly to soil fertility improvement. The influence of conservation agriculture (CA) in southern Africa on their populations has not yet been fully understood. Thus, the objective of the current study was to evaluate the effects of CA and conventional tillage on below ground biological activity in a CA long-term trial in Monze, Zambia from 2011 to 2013. The study had ten treatments which differed by tillage systems (conventional ploughing, planting basins and direct seeding) and crop diversification intensity (sole cropping, 2- or 3-year crop rotations) involving maize, cotton and sunn hemp. These factors were combined to create rotation-tillage (RotTill) treatments. Sampling of macrofauna was done once per year using a metal frame measuring 0.25 m², hand-sorted to 30-cm depth. RotTill treatments had a significant effect on earthworms (Lumbricus terrestris), termites (Coptotermes formosanus), dung beetles (Scarabaeus viettei) and centipedes (Lithobius forficatus). Earthworms and termites were more abundant in CA treatments than in conventionally ploughed (CP) treatments. Biota diversity was generally higher in CA treatments than in CP controls. Conventional mouldboard ploughing generally reduced macrofauna, thus affecting biological soil fertility and the beneficial effect of the interactions of these organisms with the soil. CA treatments had the highest maize grain yields throughout the study period. Based on the results, reduced tillage systems and crop rotations increase biological activity shown by increased densities of termites and earthworms.