Natural grasslands provide a valuable resource for livestock grazing. In many parts of South America, legume overseeding and P fertilization are commonly used to enhance primary productivity. The effect of this practice on the plant community is well established. However, how this management regime affects the soil microbiome is less known. Here, to contribute to filling this knowledge gap, we analyzed the effect of Lotus subbiflorus overseeding, together with P fertilization, on soil microbial community diversity and activity in the Uruguayan Pampa region. The results showed that plant communities in the natural grassland paddocks significantly differed from those of the managed paddocks. In contrast, neither microbial biomass and respiration nor microbial diversity was significantly affected by management, although the structure of the bacterial and fungal communities were correlated with those of the plant communities. AM Fungi relative abundance, as well as several enzyme activities, were significantly affected by management. This could have consequences for the C, N, and P content of SOM in these soils, which in turn might affect SOM degradation.