<p>Arbuscular mycorrhizal fungi (AMF) form symbiotic associations with plant roots, enhancing water and nutrient absorption. Phosphate-solubilizing fungi (PSF) can solubilize and mineralize phosphorus, an essential nutrient with low bioavailability, and eventually interact with AMF. However, the understanding of how they interact in the hyphosphere, where root influence is absent, remains limited. Furthermore, the effect of PSF on the phosphatase activity of AMF, related to the P efficiency in acquisition and utilization, within the hyphosphere and mycorrhizosphere zones, remains unclear. Therefore, this study aimed to assess the effect of three different PSF (<i>Talaromyces flavus</i>, <i>T. helicus</i>, and <i>T. diversus</i>) exudates on extracellular acid phosphatases and alkaline phosphatases associated with intra- and extraradical AMF structures in the hyphosphere and mycorrhizosphere, in vitro. To achieve this aim, the AMF <i>Rhizophagus intraradices</i> was cultured with Ri T-DNA transformed carrot roots in a system using Petri dishes that mimicked the hyphosphere (with 2 sections: (a) with roots and AMF, and (b) with only AMF) and the mycorrhizosphere (with roots and AMF in the same place). Different concentrations of PSF exudates were placed in either the hyphosphere or the mycorrhizosphere, and at the end of the experiment (8 weeks), the phosphatase activity of the AMF was measured. This research highlights that the enzymatic activity of AMF is modulated by PSF exudates, depending on whether these exudates are present in the hyphosphere or the mycorrhizosphere. Exudates in the hyphosphere, where PSF are directly associated with AMF hyphae, have a more pronounced effect on AMF extraradical alkaline phosphatases than acid phosphatases, and promote symbiosis efficiency. In contrast, PSF exudates in the mycorrhizosphere had a neutral or negative effect on symbiosis efficiency, improving the extraradical alkaline phosphatases of AMF and the acid phosphatases of the roots. Also, the effect depends on the fungal identity. AMF act as mediators in this context, improving communication between the roots and the hyphosphere microbiome. When exploring the soil, the hyphae encounter compounds produced by microorganisms, thus establishing a complex network of interactions. These interactions enhance the symbiotic efficiency of AMF, modulating the host plant without direct contact. These results show that microbial interactions not only influence the efficiency of phosphorus transfer to plants but also have broader implications for soil health and fertility management.</p> Graphical abstract <p>Effect of Phosphate Solubilizing Fungi exudates (PSF: <i>Talaromyces flavus </i>S73, <i>T. helicus</i> L7B and <i>T. diversus</i> TM) on the Arbuscular Mycorrhizal Fungi <i>(AMF: Rhizophagus intraradices GA5)</i> acid and alkaline phosphatase activity within the hyphosphere or the mycorrhizosphere. ALPm: alkaline phosphatases associated with the extraradical AMF mycelium, ALPr: alkaline phosphatases associated with the intraradical AMF mycelium, ACP: extracellular acid phosphatases</p> <p></p>