Using native seed mixtures to create or recover grassland habitats in rotation to crops or in strips surrounding fields is considered a cost-effective practice to enhance ecosystem resilience and agro-biodiversity. The aim of this research was to assess the effects of native hayseed mixtures on plant and microarthropod communities in an agricultural area of Northern Italy. Three different experimental treatments were set up. The first was a control (C) (i.e., non-seeded plots left to spontaneous vegetation succession after ploughing no deeper than 15 cm). The second, hayseed seeded (Hs) after ploughing no deeper than 15 cm. The third experimental treatment was hayseed overseeded (Ov) on the resident plant community after only a superficial harrowing. Ov plots exhibited the preeminent positive effects on the total productivity and quality of the grassland in terms of total vegetation cover, cover and richness of typical grassland species (i.e., Molinio-Arrhenatheretea species), and cover of legumes, grasses and perennial species. Moreover, Ov sites exhibited the highest abundance of microarthropod taxa and soil biological quality (QBS-ar) but only in spring, when the disturbance of ploughing negatively affected Hs and C plots. On the other hand, Hs sites showed a great reduction of invasive alien (i.e., Ambrosia artemisiifolia and Artemisia verlotiorum) and segetal weed species (i.e., Capsella bursa-pastoris and Spergula arvensis) in terms of cover. This study provides valuable indication on using hayseed mixtures to create grassland habitats as reservoir of native flora and soil biodiversity in agriculture areas.