Untreated dental caries is an important challenge in human life. In this work, the authors synthesized a new poly(vinyl alcohol) (PVA)–gum arabic (GA)–magnesium oxide (MgO) nanoparticle bionanocomposite and evaluated its antibacterial properties against Streptococcus mutansbiofilm in vitro. For optimization of variables to obtain the maximum antibacterial properties, the L9 orthogonal array of the Taguchi method was used for the design of extraction conditions. The nanocomposites were characterized by X-ray diffraction, field emission scanning electron microscopy (FESEM), energy-dispersive X-ray spectroscopy, thermogravimetric analysis and transmission electron microscopy. The FESEM images of the nanocomposite show that the nanoparticles are entirely surrounded with polymer chains of the matrix and nanoparticles are deposited on its walls; thus, a layered nanocomposite is formed. The sheet width and size ranges of the nanocomposite were determined to be between 30 and 40 and 20 and 90 nm, respectively. The results showed that the nanocomposite synthesized under conditions of 1 mg/ml PVA, 3 mg/ml GA and 6 mg/ml magnesium oxide (experiment 3) had the strongest antibacterial activity against the S. mutansbacteria’s biofilm. Under these optimal conditions, the bacterial survival rate was the lowest at 0.21 colony-forming units (CFU)/ml and the activity of S. mutansbacteria could be inhibited at a rate of 0.09 CFU/ml.