Steam-treated isomorphously substituted [Fe,Al]MFI zeolite is known to exhibit superior catalytic performance in the direct oxidation of benzene to phenol, using N2O as oxidant (BTOP). However, despite extensive efforts, the nature of the active sites in the [Fe,Al]MFI catalyst for the BTOP reaction is still largely unknown. However, recent investigations have shown strong indication that extra-framework iron species in the [Fe,Al]MFI zeolite are the catalytically active constituents in the BTOP reaction. To elucidate the active sites, this study investigates the relationship between the structure and activity of [Fe,Al]MFI catalysts in the BTOP reaction. Several isomorphously substituted [Fe,Al]MFI zeolites, varying in iron and aluminum concentrations, were successfully prepared via hydrothermal synthesis. All as-synthesized [Fe,Al]MFI zeolites were enriched with 57Fe isotope to enhance the Mauer effect, and thus providing an excellent signal-to-noise ratio. The evolution of iron from framework to extra-framework position during sequential post-activation treatments is investigated with 57Fe Mauer spectroscopy. In addition, the redox property of the extra-framework iron species formed after steam-treatment is studied by in situ Fe K-edge XANES. The catalytic performance of steam-treated [Fe,Al]MFI zeolites, with varying iron and aluminum concentrations, is evaluated in the direct oxidation of benzene to phenol, using N2O as oxidant, as well as in the N2O decomposition. From characterization and activity data, it can be inferred that (i) not all extra-framework iron species formed after steam-treatment of the [Fe,Al]MFI zeolites are active in the BTOP reaction; (ii) active iron species in BTOP reaction are preferably formed in samples with low iron concentration, while higher N2O conversions were achieved for [Fe,Al]MFI catalysts with increasing iron loading; (iii) the occurrence of catalyst deactivation, most likely due to coke formation, is more favorable in the presence of aluminum; while in the N2O decomposition, catalysts with higher aluminum concentration exhibit superior performance; and, (iv) aluminum does not play a role in the BTOP catalysis. Finally, in situ 57Fe Mauer studies show that there are several active sites for the direct oxidation of benzene to phenol. These active sites are most likely small clusters of iron species with low nuclearity (e.g. enzyme-like systems). Thus, this heterogeneous catalyst is simply heterogeneous in terms of the extra-framework iron species formed after steam-treatment, which makes it difficult to establish a direct relationship between structure and activity in the BTOP reaction.