The formation of biofilms on the surface of dental implants and abutment materials may lead to peri-implantitis and subsequent implant failure. Recently, innovative materials such as polyether-ether-ketone (PEEK) and its modifications have been used as abutment materials. However, there is limited knowledge on microbial adhesion to PEEK materials. The aim of this in vivo study was to investigate biofilm formation on the surface of conventional (titanium and zirconia) and PEEK implant abutment materials. Split specimens of titanium, zirconia, PEEK, and modified PEEK (PEEK-BioHPP) were manufactured, mounted in individual removable acrylic upper jaw splints, and worn by 20 healthy volunteers for 24 h. The surface roughness was determined using widefield confocal microscopy. Biofilm accumulation was investigated by fluorescence microscopy and quantified by imaging software. The surface roughness of the investigated materials was <0.2 µm and showed no significant differences between the materials. Zirconia showed the lowest biofilm formation, followed by titanium, PEEK, and PEEK-BioHPP. Differences were significant (p < 0.001) between the investigated materials, except for the polyether-ether-ketones. Generally, biofilm formation was significantly higher (p < 0.05) in the posterior region of the oral cavity than in the anterior region. The results of the present study show a material-dependent susceptibility to biofilm formation. The risk of developing peri-implantitis may be reduced by a specific choice of abutment material.