Objectives To test if a new biofilm-inhibiting antisense phosphorothioate oligodeoxyribonucleotide (PS-ODN) targeting simultaneously the selected homologous region of Streptococcus mutans glucosyltransferase (gtf) B, gtfC and Streptococcus sobrinus gtfI mRNAs is compatible with a sodium fluoride (NaF) mouthwash in suppressing streptococci biofilm formation in vitro. Methods Mixed cultures of S. mutans UA159 and S. sobrinus SL1 bacteria were grown anaerobically on vertical glass slides in 24-well cell culture plates containing Todd Hewitt broth with 1% sucrose and sterile-filtered human saliva (1:10) or 10% heat-inactivated horse serum under exposure to 10 µM of PS-ODN (5ʹ-GCAGACCATTGCTTAATCT-3ʹ) and 22.6 ppm of fluoride resulting from mouthwash (% w/v): 0.06% PS-ODN, 0.05% NaF, 0.1% saccharin-Na, 0.2% menthol. Untreated bacteria and bacteria exposed to mouthwash without PS-ODN, served as controls. After 24 h of incubation, glass slides were air-dried and further used for quantitative evaluation of biofilm applying optical profiler Sensofar PLµ 2300 and Gwyddion 2.27 software. Data obtained from two experiments (n=12, surface roughness; n=10, biofilm thickness) were analyzed using SPSS 23.0 program. Results The PS-ODN in combination with NaF mouthwash decreased surface roughness (parameter Rq) of the biofilm-covered glass slides by 62% and 75% versus untreated bacteria (p<0.05) in medium containing saliva and serum, respectively. This combination also reduced biofilm thickness by 88% and 78% versus untreated bacteria (p<0.05) in medium with saliva and serum, respectively. Mouthwash alone decreased roughness (parameter Rq) by 23% compared to untreated bacteria (p<0.05) growing in presence of saliva but not in serum-containing medium (p>0.05), whereas it reduced biofilm thickness by 38% and 22% versus untreated bacteria (p<0.05) in medium with saliva and serum, respectively. Conclusions The antisense PS-ODN is compatible with a NaF mouthwash and significantly inhibits mutans group streptococci biofilm formation on glass, and the combination might therefore be used as antibiofilm and/or anticaries agent in mouthwash formulations.