Purpose: This study evaluated the antibacterial activity of water-activated zinc polycarboxylate cement with adjunctive 0.12% chlorhexidine gluconate on the subgingival microbiota in fixed partial dentures. Materials and Methods: Thirty-six teeth prepared as fixed partial denture abutments in 9 patients were cemented randomly using water-activated zinc polycarboxylate cement (control group) or water-activated zinc polycarboxylate cement, including 0.12% chlorhexidine gluconate (test group). A total of 108 subgingival plaque samples were analyzed at baseline, immediately before permanent cementation (5 weeks), and 8 weeks later (at 13 weeks). Results: In the control group, the subgingival microbiota altered to closely resemble the flora of chronic gingivitis (increased proportions of gram-negative anaerobes such as Prevotella intermedia, Fusobacterium nucleatum) by 13 weeks. In contrast, the microflora at test sites comprised predominantly gram-positive facultative cocci and rods at 13 weeks. Conclusion: This study demonstrated that the addition of 0.12% chlorhexidine gluconate may enhance the antimicrobial action of polycarboxylate cements to ensure the maintenance of a microflora compatible with periodontal health, at least up to 13 weeks post-cementation.