Objective: This randomized clinical trial aimed at comparing radiological, clinical and microbial effects of surgical treatment of peri-implantitis alone or in combination with enamel matrix derivative (EMD). Methods: Twenty-six subjects were treated with open flap debridement and decontamination of the implant surfaces with gauze and saline preceding adjunctive EMD or no EMD. Bone level (BL) change was primary outcome and secondary outcomes were changes in pocket depth (PD), plaque, pus, bleeding and the microbiota of the peri-implant biofilm analyzed by the Human Oral Microbe Identification Microarray over a time period of 12 months. Results: In multivariate modelling, increased marginal BL at implant site was significantly associated with EMD, the number of osseous walls in the peri-implant bone defect and a Gram+/aerobic microbial flora, whereas reduced BL was associated with a Gram−/anaerobic microbial flora and presence of bleeding and pus, with a cross-validated predictive capacity (Q2) of 36.4%. Similar, but statistically non-significant, trends were seen for BL, PD, plaque, pus and bleeding in univariate analysis. Conclusion: Adjunctive EMD to surgical treatment of peri-implantitis was associated with prevalence of Gram+/aerobic bacteria during the follow-up period and increased marginal BL 12 months after treatment.