Clinical, microbiological, and biochemical features of human mandibular subperiosteal dental implants exhibiting peri-implantitis were compared with those experiencing long-term peri-implant health. After evaluation of clinical parameters, submucosal plaque samples were obtained from permucosal implant abutment posts exhibiting probing depths ≥5 mm and bleeding on probing in subjects with peri-implantitis (n = 3) and from posts with peri-implant health in subjects with long-term subperiosteal implant health (n = 8). The microbial specimens were transported in VMGA III and plated onto enriched Brucella blood agar and Hammond's selective medium with anaerobic incubation, and onto selective TSBV with 5% CO2 incubation. Total anaerobic viable counts and selected bacterial species were identified using established phenotypic methods and criteria. In vitro resistance to doxycycline (2 μg/mL), amoxicillin (2 μg/mL), or metronidazole (4 μg/mL) was recorded per subject when bacterial pathogen growth was noted on antibiotic-supplemented isolation plates. Interleukin (IL)-1β levels were measured with an enzyme-linked immunosorbent assay in peri-implant crevicular fluid samples from 5 study subjects. Significantly higher Plaque Index scores, higher total anaerobic viable counts, more red complex species, and lower proportions of gram-positive facultative viridans streptococci and Actinomyces species were detected on peri-implantitis-affected subperiosteal implants as compared with subperiosteal implants with long-term peri-implant health. No in vitro resistance to the 3 test antibiotic breakpoint concentrations studied was found, except a Fusobacterium nucleatum strain resistant to doxycycline at 2 μg/mL from 1 peri-implantitis subject. Subperiosteal implants with peri-implantitis tended to yield higher peri-implant crevicular fluid IL-1β levels. The level of peri-implant supramucosal plaque control and the composition of the peri-implant submucosal microbiome may be important determinants of the long-term clinical status of mandibular subperiosteal dental implants.