Purpose: To evaluate four decontamination strategies utilizing enzymatic agents available in most clinical settings to determine (1) the amount of biomaterial that can be removed in a group of previously used healing abutments (uHAs) and (2) the degree to which the decontaminated HAs are capable of inducing an inflammatory response in vitro compared to new HAs. Materials and Methods: In total, 50 HAs were collected following 2 to 4 weeks of intraoral use and distributed randomly into five test groups (groups A–E; n = 10 per group). Group A used enzymatic cleaner foam and an autoclave. Group B used an ultrasonic bath with enzymatic cleaner and an autoclave. Group C used a prophy jet, enzymatic cleaner foam, and an autoclave. Group D used a prophy jet, an ultrasonic bath with enzymatic cleaner, and an autoclave. Lastly, group E used a prophy jet and an autoclave. The control group consisted of 10 new and sterile HAs. Residual protein concentration was determined by a Micro BCA protein assay (Thermo Fisher Scientific), while HAs from each group were stained with Phloxine B and macroscopically examined for the presence of debris. To examine the inflammatory potential, human primary macrophages were exposed to HAs, and supernatant levels of nine cytokine and chemokine profiles were analyzed using a multiplex bead assay. Results: All test groups showed differences in the degree of visual decontamination compared to controls. Groups D and E displayed the most effective surface debris removal and reduced protein concentration, while group A was the least effective. However, compared to controls, all test groups showed high levels of inflammatory cytokine secretion via multiplex assay for up to 5 days. Conclusions: Our study found that decontamination of uHAs utilizing enzymatic cleaners failed to reestablish inert HA surfaces and prevent an inflammatory immune response in vitro. Clinicians should not reuse HAs even after attempts to decontaminate and sterilize HA surfaces.