Although chickens can improve the chemical properties of soil through the deposition of excreta, their effects on soil macrofauna are poorly known. This work assessed the effects of grazing indigenous laying hens on soil macrofauna of two agroecosystems in Portugal: an organic horticultural field and a conventional orchard. At the horticultural field, laying hens were used to control weeds and the results were compared with those of two other weed control treatments: mechanical (rototiller) and thermal (flame weeding). At the orchard, the effects of hens were compared to that of the orchard understory vegetation, as a control. Soil epigeic macrofauna was collected in both locations, and earthworms were only collected in the horticultural field. Relative to the other treatments, grazing in the horticultural field increased the density of earthworms in the medium term (ranging from 150 to 625 earthworms/m2), without harming the density and diversity of epigeic macrofauna. However, at the orchard, the grazed soils presented lower soil epigeic macrofauna diversity, as well as significantly lower density of spiders than the control (4.67 vs 8.67 individuals/sample, respectively). These results suggest that the grazing effects can be affected by several factors, including the type of agroecosystem and farm management. Further research is required to optimize grazing management in different farming systems, considering animal density and grazing duration, thus ensuring the best contributions of chickens to soil fertility.