The objective of this research was to isolate and identify native communities and perform individual isolates of arbuscular mycorrhizal fungi (AMF) from an experimental field with diversity of symbiont fungi, located near San Piero a Grado (Pisa), in the northwest of Tuscany, a natural reserve on the east coast of Italy. The isolation and identification procedures of native AMF obtained from trap cultures established with soils collected from different experimental sites are describes. A total of 36 trap cultures were prepared using various host plants, including Zea mays, Petroselinum crispum and spontaneous species emerging from the native soil seed bank. Several morphotypes were propagated and subsequently characterized through morphological and molecular analyses. The isolates were identified as Funneliformis mosseae, Claroideoglomus etunicatum, Racocetra fulgida, Diversispora aurea, Funneliformis geosporum, and Septoglomus constrictum. These isolates were preserved in pure cultures to obtain spores for functional characterization. The study of native communities revealed the impact of local AMF species richness.