Chronic Periodontitis (CP) is a disease characterized by interactions between microbial pathogens and host immune response that have a crucial role in its initiation and progression via the release of inflammatory mediators such as tumor necrosis factor- α (TNF-α). Pentraxin-3 (PTX3), the first long pentraxin described, is produced in response to pro-inflammatory cytokines by a variety of cells including those abundant in periodontal tissues. The aim of the present study was to investigate the role of PTX3 compared to TNF -α as a diagnostic and prognostic marker for CP. Gingival Crevicular Fluid (GCF) samples were taken from 53 CP patients before and after scaling and root planing (SRP) as well as from 50 periodontally healthy subjects as a control group. The GCF samples were tested for PTX3 and TNF-α levels by Enzyme-Linked Immunosorbent Assay (ELISA). Differences between CP patients and healthy subjects regarding clinical parameters and tested biomarkers in GCF were assessed. Moreover, the correlations between them were calculated. Out of 465 female patients examined, 53 patients were diagnosed as CP with a prevalence rate of 11.4%. The mean values of periodontal parameters were significantly reduced after treatment. Mean TNF-α and PTX3 levels were higher in CP patients (935.27 ± 264.21) (2.49 ± 0.537) than in the healthy group (772.32 ±0.148) (1.077 ± 0.084) respectively, with a significant difference between both groups (p < 0.05). Furthermore, these levels were reduced after treatment with a highly statistically significant difference. Positive correlations between the mean values of TNF-α, PTX3 levels, and clinical parameters were found whereas, PTX3 was more positively correlated. In conclusion, both PTX3 and TNF-α in GCF, previously recommended to be used as diagnostic markers for CP, can be also used as prognostic markers for follow-up of CP patients in addition to clinical and radiological parameters.  However, further large-scale studies on both genders are recommended to confirm their role as prognostic markers.