We present a method to localise and quantify surface-bound phosphatase activity (SBPA) in mycorrhizal fungi and mycorrhizae of Nothofagus obliqua (Mirb.) Oerst., using image processed confocal fluorescent microscopy. ELF-97 is a hydrophilic substrate which turns into a strongly fluorescent precipitate upon activation by phosphomonoesterases. In fungal mycelium, this technique has recently been approved by comparison with a standard method (p-nitrophenyl-phosphate). The microscopic technique based on ELF-97 revealed that Paxillus involutus (Batsch: Fr.) Sing. and Austropaxillus boletinoides (Sing.) Brsky. & Jarosch provide different adaptive strategies to changing phosphate concentrations and different pH (3-7). We also analysed SBPA in four mycorrhizal associations of N. obliqua and found that the organisation of the mantle played an essential role regarding the SBPA. In general, mycorrhizal roots shifted SBPA from the root to the mantle. In this context, Pisolithus tinctorius (Pers.) Coker & Couch proved to be the most relevant mycorrhiza partner for N. obliqua by increasing significantly the overall SBPA of the mycorrhiza in respect to non-mycorrhizal roots. In conclusion, ELF-97 fluorescence microscopy in combination with image processing routines determined SBPA and revealed phosphorus (P) adaptation strategies of mycorrhizal fungi and mycorrhizae on a structural-physiological level