Hyaluronic acid in its various forms shows bacteriostatic, fungistatic, anti-inflammatory, anti-edematous, osteoinductive, and pro-angiogenetic properties. This study aimed to evaluate the effect of subgingival delivery of 0.8% hyaluronic acid (HA) gel on clinical periodontal parameters, pro-inflammatory cytokines (IL-1 beta and TNF-alpha) and biochemical markers of inflammation (C-reactive protein (CRP) and alkaline phosphatase (ALP) enzymes) in patients with periodontitis. Seventy-five patients with chronic periodontitis were divided randomly into three groups (25 in each group): group I received scaling and surface root debridement (SRD) + HA gel; group II received SRD + chlorhexidine gel; and group III received surface root debridement alone. Clinical periodontal parameter measurements and blood samples were collected to estimate pro-inflammatory and biochemical parameters at the baseline before therapy and after two months of therapy. The results show that HA gel has a significant effect on the reduction in clinical periodontal parameters (PI, GI, BOP, PPD, and CAL), IL-1 beta, TNF-alpha, CRP, and ALP after 2 months of therapy as compared to the baseline (p < 0.05) with nonsignificant differences from the CHX group (p > 0.05), except GI (p < 0.05), and significant differences from the SRD group (p < 0.05). Moreover, significant differences were found between the three groups regarding the mean improvements of GI, BOP, PPD, IL-1β, CRP, and ALP. It can be concluded that HA gel has a positive effect on clinical periodontal parameters and improvements in inflammatory mediators similar to chlorhexidine. Therefore, HA gel can be used as an adjuvant to SRD in the treatment of periodontitis.