This study investigated subgingival peri-implant biofilm formation on four luting agents (Kerr TempBond, Harvard Dental Harvard Cement, 3M ESPE RelyX Unicem, and Kuraray Panavia F 2.0) under realistic in situ conditions. Samples of the luting agents were positioned in the subgingival area of healing abutments, and the biofilm accumulation on the samples at the interface between luting agent and titanium and on the smooth titanium surface was investigated using scanning electron microscopy. In comparison to plane titanium surfaces, interfaces between implant abutment, cement, and suprastructure showed an increased bacterial accumulation and should therefore be regarded as predisposing substrates for peri-implant biofilm formation.