Milk fat globule and membrane (MFGs/MFGM) ingredients are the main source of the phospholipid supplementation in the dairy industry. Their physicochemical properties and functionalities play important roles in their application. This study investigated the effect of acidification (pH 6.30 and pH 5.30) on the microstructure, interfacial and thermal properties of MFGs/MFGM sourced from bovine milk (control pH 6.70). Meanwhile, sweet whey was prepared to mimic the raw material used in the industrial production of MFGs/MFGM (MFGM-C). The study revealed a broad particle size distribution of MFGs/MFGM at pH 5.30, while smaller particles were found in MFGM-C. This was confirmed by confocal scanning microscopy (CLSM) images, which showed that few intact MFGs existed in the MFGM-C. Fourier Transform Infrared Spectroscopy (FTIR) indicated more peaks belonging to protein-lipids associations presented at pH 6.30. Additionally, the interfacial tension of MFGs/MFGM at pH 6.30 was significantly lower than at pH 6.70 and 5.30. X-ray Diffraction Spectra (XRD) showed that the fat in MFGM-C exhibited no detectable crystalline structures, and fewer α and β′ crystals appeared at pH 6.30. All MFGs/MFGM from bovine milk or whey exhibited both exothermic and endothermal events. The smallest heat enthalpy of crystallization (ΔHC) and melting (ΔHM) was observed in MFGM-C, followed by those at pH 6.30. Compared to MFGs/MFGM at pH 6.70, pH 6.30 induced more profound effects on the interfacial and thermal properties of MFGs/MFGM than pH 5.30. Acidification and the use of different raw materials lead to differentiation in the physicochemical and techno-functionalities of MFGs/MFGM.