Background: Allografts and polyetheretherketone (PEEK) cages are the two most commonly used material in anterior cervical discectomy and fusion (ACDF). However, their effectiveness in in two-level ACDF remains controversial. The primary aim of this retrospective study is to compare the clinical and radiological outcomes of two-level ACDF with plate fixation using either a structural allograft or a PEEK cage. Methods: From 2010 to 2015, 88 consecutive patients underwent a two-level ACDF of which 53 used an allograft and 35 patients with a PEEK cage. All PEEK cages were filled with local autografts. All clinical outcomes were prospectively collected preoperatively, at six months and at two years after surgery. Clinical efficacy was evaluated using visual analogue scale for neck pain and limb pain, the Neck Pain and Disability Score, Neck Disability Index, Neurogenic Symptom Score, and the Japan Orthopedic Association score. Radiological outcomes were assessed preoperatively, immediately after surgery, and at the final follow-up. Results: A preoperative comparison revealed no difference between the two patient groups in terms of age, gender, body mass index, smoking status, preoperative symptoms, operated levels, and follow-up (mean= 42.8 months). No difference in improvements in the clinical outcome between the two groups was observed. Both groups showed significant improvement in mean disc height, segmental height, and segmental lordosis postoperatively. The fusion rates for PEEK cage was 100% at both levels while the fusion rates for allograft group was 98.1% at cephalad level and 94.2% at caudad level (p>0.05). Subsidence at the cephalad level occurred in 22.9% (8/35) segments in the PEEK group and 7.7% (4/52) segments in the allograft group (p=0.057). At the caudal level, a higher cage subsidence was noted in the PEEK group compared to the allograft group [37.1% (13/35) versus 15.4% (8/52)] (p=0.02). Overall, subsidence was noted in 30% (21/70) of the PEEK group and in 11% (12/104) of the allograft group (p<0.05). Conclusion: The use of PEEK cages resulted in a higher rate of subsidence in two-level ACDF as compared to allograft. Two-level ACDF using either allografts or PEEK cages resulted in similar clinical outcomes, radiological improvements in alignment and fusion rates.