Monolayers were prepared from phospholipids extracted from bovine milk and used as a model system to mimic the native milk fat globule membrane (MFGM) surface structure in various microscopic experiments. The natural complex mixtures of phospholipids were isolated from bovine raw milk, raw cream, processed whole milk, and buttermilk powder by total lipid extraction and solid-phase extraction. A Langmuir film balance mounted on an epifluorescence microscope was used to analyze the physical behavior of the monolayer films and the phase coexistence resulting from the formation of phospholipid microdomains within these films. Atomic force microscopy was used for nanometer-scale topographic resolution of the microdomains. This study allowed comparison of the behavior of phospholipid monolayers from dairy products at different stages of processing, analysis of the formation of microdomains, and the study of the effect of milk processing on lipid-lipid interactions and phase coexistence. It was observed that milk processing changes the physical behavior of phospholipid monolayers by altering the phospholipid profile and the fatty acid distribution.