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Objective: The high risk of nonunion represents a challenge in vertebral surgery, thus stimulating new strategies to improve fusion rates. We investigated the effect of 2 different bone grafts and amniotic fluid application on radiologically and histologically evaluated vertebral fusion in an experimental rat model. Materials and methods: Forty-eight 24-week-old Sprague Dawley rats were included and assigned into 1 of 4 groups: allograft group, allograft plus human amniotic fluid group, demineralized bone matrix (DBM) group, or DBM plus human amniotic fluid group. After decortication and L4–L6 spinal fusion, study treatments were applied. Fusion in each rat was examined radiologically and histologically 8 weeks after the intervention. Results: The group that received only allograft had better radiologic scores (median = 3.5; range = 3–4) when compared with the group that received only DBM (median = 2; range = 1–4) (P = 0.002); however, histologic scores did not differ. When amniotic fluid was added to the grafting, allograft-based treatments performed better than DBM-based treatments both on radiologic (median = 4; range = 3–4 vs median = 3; range = 3–4; P = 0.003) and histologic (median = 7; range = 6–7 vs median = 5; range = 3–6; P < 0.001) evaluation. Addition of amniotic fluid did not result in better outcomes in the rats that received DBM-based treatments but based on histologic evaluation, rats that received allograft-based treatments benefited from this application. Conclusions: Amniotic fluid seems to have an enhancing effect on posterior spinal fusion, particularly when combined with allograft.