Biotite crystals are phyllosilicate trioctahedral micas with the general chemical formula K(Mg,Fe)₃AlSi₃O₁₀(OH)₂ that form a solid-solution series with iron-poor phlogopite and iron-rich annite endmembers. With a wide band gap energy and a layered structure with free surface charges, biotite nanosheets can be readily obtained by cleavage methods and used as dielectrics in nanodevice fabrication for the next generation of electronics and energy harvesting. Here, a comprehensive study of biotite samples with different iron concentrations and oxidation states is presented. Structural, optical, magneto-optical, and magnetic characterizations were performed using several experimental techniques, including state-of-the-art synchrotron-based techniques, to correlate the iron chemistry (content and oxidation state) with the macroscopic properties of both minerals. The study reveals a nanoscale-homogeneous Fe distribution via synchrotron X-ray fluorescence mapping, defect-mediated optical transitions modulated by Fe³⁺/Fe²⁺ ratios, and temperature-dependent magnetic transitions from paramagnetism to competing ferro−/antiferromagnetic interactions. Furthermore, the use of these biotite crystals as substrates for ultrathin heterostructures incorporating monolayer (ML) MoSe₂ is explored by magneto photoluminescence at cryogenic temperatures. The results show that the presence of iron impurities in different oxidation states significantly impacts the valley properties for ML-MoSe₂. Overall, these findings offer a comprehensive interpretation of the physical properties of bulk biotites in a correlative approach, serving as a robust reference for future studies aiming to explore biotites in their ultrathin form.