Minerals in groundwater interact with various chemical and biological species including organic matter, proteins, and prevalent oxyanions, resulting in surface coatings and thin films of these different components. Surface interactions and the surface adsorption of these components on both oxide and oxyhydroxide iron surfaces have been widely investigated using a variety of spectroscopic methods. Despite these numerous studies, there still remains uncertainty with respect to interactions between these individual components, as well as heterogeneities and phase segregations within these thin films. In this study, we investigate mixtures containing Fe-containing minerals, proteins, and oxyanions to better understand surface interactions and phase segregation using Atomic Force Microscopy PhotoThermal Infrared (AFM-PTIR) spectroscopy. The results of this study show that AFM-PTIR spectroscopy can identify both nano- and microscale heterogeneities present within these thin films that are difficult to discern with other more conventional techniques such as ATR-FTIR spectroscopy due to phase segregation and mineral surface interactions. Overall, AFM-PTIR spectroscopy provides insights into multi-component environmental films that are difficult to uncover using other methodologies. This method has the potential to differentiate between bound and unbound toxic species as well as biological components, including environmental DNA, which can be used to assess the fate and transport of these species in the environment.