Background: Shrub encroachment (SE) is a phenomenon in which grasses and herbaceous vegetation are replaced by woody shrubs. The progressive spread of shrubs represents a form of land cover change that is widespread in arid and semi-arid grassland ecosystems. Many previous studies have highlighted the effects of SE on soil respiration rates and nutrient storage, but little is known about its belowground effects. While previous work considered shrubs to be non-species specific or as a single intervening species, we selected an Ampelodemsos mauritanicus grassland and six coexisting shrubs (i.e. Pistacia lentiscus L., Juniperus phoenicea L., Myrtus communis L., Rosmarinus officinalis L., Olea europaea L., and Euphorbia dendroides L.) to investigate the effects of their encroachment on soil microbiota. We used high-throughput sequencing, coupled with soil chemical analyses and litter using 13C CPMAS NMR spectroscopy.Results: Results showed a strong influence of shrub canopy on bacterial and fungal community diversity, species richness and overall community composition in the soil. Litter chemistry was dominated by O-alkyl-C, with the highest content in Ampelodesmsos and Euphorbia, but richer of aromatic C in Pistacia and Rosmarinus. Bacterial diversity was highest under Juniperus and Euphorbia, while lowest under Rosmarinus and grassland. Conversely, fungal diversity was highest under Olea and Euphorbia, while lowest under Myrtus and grassland. Moreover, soil C and N contents were highest under Olea, Pistacia and Myrtus compared to the other canopies. In addition, grassland and Rosmarinus had the highest Fe content. Furthermore, increased co-occurrence network size and connectivity were recorded under shrubs compared to the grassland matrix.Conclusions: Our results suggest that the individual effect of each shrub on the grassland matrix depends mainly on the chemical properties of the shrub litter, which alters the chemical profile of the soil and, in cascade, shapes the associated microbiota.