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With the increased incidence of traumatic injuries and the advanced understanding of the periodontal and alveolar healing process, teeth splinting has become a common practice for stabilizing traumatized teeth. Consequently, several splinting materials and techniques have been introduced in the past few years. Despite the detrimental role of bacterial biofilm on healing, the level of biofilm development on these material surfaces has not been well investigated. Bacterial biofilms are severely detrimental for periodontal healing of avulsed and luxated teeth. Thus, biofilm growth becomes a critical factor in selecting the material of choice for dental splints. In this study, we aim to assess the level of oral biofilm growth on four different splinting systems: Ribbond©, orthodontic NiTi wire, monofilament fishing line, and Titanium Trauma Splint. A total of 72 extracted anterior teeth were divided into four groups. We splinted six rows of three teeth each per group. The teeth selected were caries-free and periodontitis-free at the time of extraction. To assess biofilm growth, a supragingival dental plaque sample was cultured and directly inoculated into all groups. After 7 days, bacterial growth was quantified by live/dead fluorescent microscopy assay and colony forming unit counts (CFU). Using one-way ANOVA and Bonferroni’s post hoc tests, we demonstrated that all splint systems allowed for bacterial growth. However, the Titanium Trauma Splint (TTS) allowed for the least amount of biofilm growth compared to other splint systems.