The objective of this study was to develop novel modified giomers by incorporating the antibacterial quaternary ammonium monomers (QAMs), dimethylaminododecyl methacrylate (DMADDM) or dimethylaminohexadecyl methacrylate (DMAHDM) into a commercial giomer. The material performances including mechanical properties, surface characteristics, color data, cytotoxicity and fluoride release of the novel giomers were evaluated. Antibacterial activity against severe early childhood caries (S-ECC) saliva-derived biofilms was assessed by lactic acid production measurement, MTT assay, biofilm staining and 16S rRNA sequencing. A rat model was developed and the anti-caries effect was investigated by micro-CT scanning and modified Keyes’ scoring. The results showed that the material properties of the QAMs groups were comparable to those of the control group. The novel giomers significantly inhibited lactic acid production and biofilm viability of S-ECC saliva-derived biofilms. Furthermore, caries-related genera such as Streptococcus and Lactobacillus reduced in QAMs groups, which showed their potential to change the microbial compositions. In the rat model, lesion depth, mineral loss and scoring of the QAMs groups were significantly reduced, without side effects on oral tissues. In conclusion, the novel giomers incorporated with antibacterial QAMs could inhibit the cariogenic biofilms and help prevent secondary caries, with great potential for future application in restorative treatment.