<p>Chlorhexidine and antibiotics are commonly used as adjunct treatments for periodontitis. However, these antimicrobials can lead to microbial resistance and chlorhexidine can impair health-associated nitrate (NO₃^-) metabolism. We tested the effect of chlorhexidine (0.002%), metronidazole (16&#xa0;µg/ml) and amoxicillin (0.7&#xa0;µg/ml), with and without 8 mM NO₃^-, on the bacterial composition and NO₃^- metabolism of subgingival plaque samples from 12 periodontitis patients grown in vitro for 8&#xa0;h. The low sublethal concentrations of amoxicillin and chlorhexidine significantly inhibited microbial growth and impaired NO₃^- reduction, whereas the physiological concentration of metronidazole did not. A lower subgingival microbial dysbiosis index (SMDI) was found in the NO₃^- condition compared with amoxicillin alone and chlorhexidine with or without NO₃^- (<i>p</i> &lt; 0.05). The SMDI of the metronidazole conditions was also significantly lower than in those with chlorhexidine (<i>p</i> &lt; 0.05). Moreover, NO₃^- alone or combined with metronidazole appeared to increase <i>Neisseria</i> spp. and <i>Aggregatibacter</i> spp., whilst disease-associated changes were found in the chlorhexidine and amoxicillin conditions. Adding NO₃^- to metronidazole led to health-associated changes compared with metronidazole alone. In conclusion, low levels of amoxicillin and chlorhexidine limited microbial growth, impaired NO₃^- metabolism and were linked to disease-associated microbial profiles. A dual treatment of metronidazole + NO₃^- should be further investigated in clinical studies.</p>