Background: The aim of this study was to evaluate and compare the bacterial colonization, cytotoxicity, immune response, and clinical parameters of three different suture materials: multifilament silk (Silk^®), monofilament nylon (Daclon^®), and expanded polytetrafluoroethylene monofilament (PTFE^®), in surgical extractions of impacted mandibular third molars. Methods: This randomized controlled clinical trial was conducted on twenty-one patients requiring surgical extraction of an impacted third mandibular molar. A bayonet-shaped flap was sutured using all three materials in each patient. Bacterial cell counting and qPCR were assessed for microbiological analysis. In vitro cytotoxicity was studied with the metabolic activity WST-1 assay. Inflammatory response was evaluated through histological analysis. Clinical parameters—healing, handling, slack, pain, swelling and trimus—were recorded. Statistical significance was set at p ≤ 0.05. Results: Monofilament sutures accumulated fewer bacteria and DNA copies than Silk^® (p < 0.05). The WST-1 assay revealed non-cytotoxic effects. Silk^® presented an immune response with lymphocyte-like cells. The highest values of pain and inflammation were reached at 48 h, with a significant correlation between them (p < 0.05). Silk and nylon were more manageable than PTFE (p < 0.001), and nylon had less slack (p < 0.001). Conclusions: Silk showed the poorest microbiological and histological performance, with higher levels of bacterial colonization and a more pronounced inflammatory response compared to the other types of suture. Clinically, it offered better handling than PTFE (PTFE^®), comparable to nylon (Daclon^®), but it exhibited greater slack, which could prove less favorable for wound stability. None of the sutures showed in vitro cytotoxicity. Monofilament sutures, particularly nylon (Daclon^®), showed better outcomes, acceptable handling, less bacterial colonization, and a milder inflammatory response.