Background The aim of the present study was to assess and quantify cluster of differentiation 163 (CD163) protein levels and CD163 messenger RNA (mRNA) gene expression in subgingival plaque samples of generalized chronic periodontitis subjects with and without type II diabetes mellitus (DM). Materials and methods Eighty chronic periodontitis subjects were selected and divided into 40 systemically healthy, generalized chronic periodontitis subjects (Group I) and 40 generalized chronic periodontitis subjects diagnosed with type II diabetes mellitus (Group II). Age, body mass index (BMI), income, plaque index (PI), bleeding on probing (BOP), probing pocket depth (PPD), and clinical attachment level (CAL) were recorded. CD163 protein and gene expressions were quantified and compared between the groups. Results The mean age, BMI, income, PI, BOP %, and CD163 protein and gene expression were higher in Group II (p< 0.05) as compared to Group I. In Group I, CD163 protein levels showed a negative correlation with respect to BMI and PI, and this was statistically significant. In Group II, all the periodontal parameters showed a positive correlation with CD163 protein levels. Overall, PI and BOP % were significantly correlated with CD163 protein levels. Both CD163 protein and gene expression showed a negative correlation with each other (p= 0.001). Conclusion The elevated protein levels of CD163 in the subgingival plaque samples of generalized chronic periodontitis individuals with type II diabetes mellitus signify the involvement of CD163 in the pathogenesis of both periodontitis and diabetes mellitus. CD163 can play a challenging role as a diagnostic, as well as a prognostic biomarker, in both these inflammatory diseases.