Effective production of oil from carbonate reservoirs often requires the application of improved oil recovery technologies such as waterflooding. However, conventional waterflooding in carbonates usually results in low hydrocarbon recovery as most of these formations exhibit a complex pore throats structure and are mostly oil-wet. Therefore, improved insight into the causes of hydrophobic wetting behavior of such reservoirs is important for understanding the fluid distribution, displacement and enhancing recovery processes. The characterization of fluid-rock interactions is, however, challenging with existing laboratory methods, which are typically based on macroscale (mm) observations. In this experimental study, an advanced imaging technique, namely environmental scanning electron microscope, was applied for the comprehensive investigation of microscale (µm) wettability variations in carbonate rocks covered with organic layers. For the first time, the presence of organic layers on the sample was proved using energy dispersive X-ray mapping. Furthermore, the chemical bond of this layer and carbonate rock surfaces was determined using the transmission electron microscopy and electron energy-loss spectroscopy. The thickness of layer was estimated by using image processing software. These findings show that the application of combined microscopic techniques reveals important details about the reason of hydrophobic wetting properties of real carbonate rocks.