Periodontal disease (PD) is an inflammatory condition of the tissues supporting the teeth, which is widespread among the adult population. Evidence shows a relationship between PD and vitamin D levels, which is involved in the regulation of bone metabolism, mineral homeostasis, and inflammatory response. This study aimed to perform a simultaneous evaluation of inflammatory mediators and vitamin D levels in saliva in periodontopathic patients to better understand their role in periodontal disease. In this observational study, clinical periodontal parameter examination was performed for each patient. Moreover, the saliva levels of 25(OH)D3, TGFβ, IL-35, IL-17A, and MMP9 were evaluated using an ELISA assay. An increase in TGFβ, IL-35, MMP9, and IL-17A salivary levels and a reduction in 25(OH)D3 levels were observed in periodontopathic patients with respect to the healthy controls. The present study revealed significant positive correlation between cytokines and highly negative correlation between 25(OH)D3 and salivary cytokine levels. Further studies are needed to better understand if salivary cytokines and vitamin D evaluation may represent a new approach for detection and prevention of progressive diseases, such as PD.