Objective: The aim of this study was to: (1) evaluate the anti-inflammatory effects of cannabidiol (CBD) on primary cultures of human gingival fibroblasts (HGFs) and (2) to clinically monitor the effect of CBD in subjects with periodontitis. Background: The use of phytocannabinoids is a new approach in the treatment of widely prevalent periodontal disease. Materials and Methods: Cannabinoid receptors were analyzed by western blot and interleukin production detected using enzyme immunoassay. Activation of the Nrf2 pathway was studied via monitoring the mRNA level of heme oxygenase-1. Antimicrobial effects were determined by standard microdilution and 16S rRNA screening. In the clinical part, a placebo-control double-blind randomized study was conducted (56 days) in three groups (n = 90) using dental gel without CBD (group A) and with 1% (w/w) CBD (group B) and corresponding toothpaste (group A - no CBD, group B - with CBD) for home use to maintain oral health. Group C used dental gel containing 1% chlorhexidine digluconate (active comparator) and toothpaste without CBD. Results: Human gingival fibroblasts were confirmed to express the cannabinoid receptor CB2. Lipopolysaccharide-induced cells exhibited increased production of pro-inflammatory IL-6 and IL-8, with deceasing levels upon exposure to CBD. CBD also exhibited antimicrobial activities against Porphyromonas gingivalis, with an MIC of 1.5 mu g/mL. Activation of the Nrf2 pathway was also demonstrated. In the clinical part, statistically significant improvement was found for the gingival, gingival bleeding, and modified gingival indices between placebo group A and CBD group B after 56 days. Conclusions: Cannabidiol reduced inflammation and the growth of selected periodontal pathogenic bacteria. The clinical trial demonstrated a statistically significant improvement after CBD application. No adverse effects of CBD were reported by patients or observed upon clinical examination during the study. The results are a promising basis for a more comprehensive investigation of the application of non-psychotropic cannabinoids in dentistry.