Octenidine-containing antiseptic oromucosal solution has proven to be efficacious and safe for temporary bacterial reduction and plaque inhibition in the oral cavity. While its clinical outcomes are encouraging, further research is needed to assess its microbicidal activity against pathogens causing gingivitis and periodontitis. We evaluated the in vitro microbicidal activity of different dilution concentrations of a commercially-available 0.1% octenidine dihydrochloride containing oromucosal solution (OOS) against 10 periodontopathogenic bacteria and three yeast strains using the testing methodology frameworks of DIN EN 13727:2012+A2:2015 (30-s contact time) and DIN EN 13624:2022-08 (60-s contact time), respectively. The OOS showed significant bactericidal activity at 80% dilution concentration, as indicated by a log reduction factor (lg RF) ≥5 for all 10 bacteria. At 50% dilution concentration, bactericidal activity was observed against specific bacteria like Porphyromonas gingivalis, Prevotella buccalis, Parvimonas micra, Eikenella corrodens, Dialister pneumosintes, Schaalia odontolytica (Actinomyces odontolyticus), and Campylobacter rectus. Yeasticidal activity against Candida albicans was observed at 80% dilution concentration, whereas Candida auris was more susceptible to the OOS and showed lg RF ≥4 even at 50% dilution concentration. Therefore, the OOS may be an effective adjunct to periodontal therapy aimed at reducing pathogenic microbial load, inhibiting plaque formation, and thereby preventing periodontal inflammation.