Although most transmission electron microscope (TEM) investigations were carried out using conventional ultramicrotomy, they were limited to the tooth/adhesive resin interface and were difficult to accomplish for the resin-composite interface. Some of these limitations have been overcome with the introduction of focused ion beam (FIB) milling. Therefore, the objective of the study was to compare different composites/adhesive interfaces using FIB/TEM technique. Cylindrical cavities were prepared in extracted human molar teeth. The restored cavities were divided into four groups: (1) One-step self-etch Scotchbond Universal (SBU; 3M ESPE, USA), (2) all-in-one Xeno-V+ adhesive (X5P; DENTSPLY, Germany), (3) two-step etch-and-rinse Prime and Bond NT (PNT; DENTSPLY, Germany) that were restored with Filtek Supreme Ultra Universal composite (3M ESPE, USA), and (4) teeth restored with the two-step self-etch Filtek Silorane adhesive (SSA; 3M ESPE, USA) that were restored with its corresponding Filtek Silorane composite (3M ESPE, USA). All specimens were cross-sectioned and subjected to FIB preparation followed by composite/adhesive interfacial TEM examination. The TEM findings were variable and ranged from concentrated clusters of nanoparticles to phase separation of adhesive components and large osmotic blisters. The osmotic blisters in all-in-one adhesives appeared to be influenced by the presence of water and the solvent's vapor pressure in the cavity. In conclusion, FIB/TEM is a powerful tool allowing the study of biomaterials interaction in situ. The absence of residual water in the adhesives may reduce osmotic blistering that, in turn, may improve the reactivity of the resin monomers, as well as interfacial bonding.