In vivo biofilm models with multi-species biofilms offer the opportunity to evaluate materials in simulated clinical conditions including composite plaque, salivary pellicle, and removal forces [18]. Although the understanding of oral biofilms and the influence of surface characteristics on microbial accumulation has increased, significant gaps in the fundamental knowledge about the formation and establishment of such microbial communities still exist. Furthermore, the most essential processes in oral biofilm formation are not yet fully understood [52]. Therefore, it is necessary to examine the correlation between bacterial adhesion—including differences between different species—and modifications of surface characteristics in simplified, reproducible, and manageable in vitro systems to transfer the knowledge on fundamental in vitro matters to new clinical biomaterial implementations. Additionally, we indicated in a previous study the possibility of a correlation between in vivo and semi-static in vitro findings in respect to microbial adhesion on surfaces with different surface properties [25]. Even in a simplified in vitro setting, the quantity and quality of bacterial accumulation are influenced by many factors; in vitro relationships between surface characteristics and bacterial adhesion depend on experimental conditions, such as preconditioning protein films and the simulation of shear forces [8, 53]. For example, salivary proteins mediate the initial accumulation of microorganisms in the human oral cavity [54]. For simulating the influence of the salivary pellicle in vitro, specimens may be incubated in various saliva solutions before bacterial adhesion testing. In the present study, all specimens were pre-incubated with artificial saliva [2], which was chosen to exclude the influence of inter-individual variations in salivary protein content and the composition of human saliva so that reproducible results could be achieved [26, 55]. Two different single-species biofilms, S. epidermidis and S. sanguinis, were used as test microorganisms to investigate the potential of differently treated implant surfaces to adhere these bacteria. S. epidermidis and S. sanguinis are not usually associated with active peri-implantitis, but they are amongst the main early colonizers of oral tissues and artificial biomaterials, paving the way for more pathogenic species [56,57,58]. S. epidermidis and S. sanguinis represent two dominant but very different bacterial families, i.e., Streptococcaceae and Staphylococcaceae, which are members of the human oral microbiome; these bacteria normally reside on the mucous membranes of humans and can bind to hard surfaces in the oral cavity [57]. S. sanguinuis is commonly present in the human oral cavity and known as a pioneer bacterium of oral biofilms [10, 18, 56, 58, 59]. S. epidermidis, normally a commensal bacterium of the skin, is a major concern for patients with surgical implants, causing the growth of pathogenic biofilms on various implant devices, such as breast and hip implants, which may result in implant failure [60]. In some recent studies, S. epidermidis has also been detected in pathogenic biofilms on failing dental implants [43]. Fluorometric techniques offer the opportunity to quantitatively investigate a high number of specimens in a short period of time and, at the same time, provide reproducible and significant data [25]. In this study, the CytoX-Violet Cell Proliferation Assay Kit was used to simply measure the amount of viable bacteria adhering to the test specimens. The fluorometric change of the indicator solution shows the activity of the cellular dehydrogenases and is directly proportional to the cell viability of adhering bacteria. It should be mentioned that this specific method fails to indicate vital adhering bacteria and cannot differentiate between cultivable vital cells and non-cultivable vital cells. This is important because large amounts of dead bacteria (up to 40%) have already been found after short incubation times [25].