The combination of osseous graft with barrier membrane and enamel matrix protein derivative (EMD) has the potential to result in a synergistic effect. Therefore, the aim of this study was to evaluate the effectiveness of EMD in combination with demineralized freeze-dried bone allograft (BG) and bioresorbable membrane (Biomesh) in the treatment of human mandibular Class II furcation defects over a period of 12 months. Thirty patients with chronic periodontitis and a single Class II furcation defect on the buccal or lingual surface of mandibular teeth were included. The clinical parameters evaluated were probing pocket depth (PPD), horizontal probing depth (HPD), vertical relative attachment level (V-RAL), and relative gingival margin level (RGML). three groups were created based on treatment method: EMD + BG + guided tissue regeneration (GTR), BG + GTR, and open flap debridement (OFD). All three groups showed a statistically significant PPD reduction of 1.74 ± 1.00 mm, 0.81 ± 0.31 mm, and 0.46 ± 0.52 mm at 12 months postsurgery. EMD + BG + GTR showed a significantly greater PPD reduction compared with BG + GTR, as well as OFD. EMD + BG + GTR showed a statistically significant vertical clinical attachment gain of 2.12 ± 1.07 mm at 12 months compared with BG + GTR as well as OFD. Significant reductions in mean HPD were observed for EMD + BG + GTR (2.10 mm) as well as BG + GTR (1.5 mm). The number of Class II furcation defects that closed or converted to Class I was greatest for EMD + BG + GTR. It can be concluded that EMD + BG + GTR resulted in a statistically significant reduction of PPD, V-RAL gain, and a nonsignificantly greater reduction of HPD compared to BG + GTR.