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Quantifying the ex vivo growth of complex multispecies dental biofilms using cross-polarization 1310-nm optical coherence tomography (CP-OCT) system was investigated.Bacterial microcosms, which were derived from plaque samples of paediatric subjects, were incubated in a biofilm reactor system containing discs of different dental materials for 72 h with daily sucrose pulsing (5×). CP-OCT analysis of biofilm mass was validated with crystal violet (CV) assays at various growth stages of these complex biofilms. CP-OCT was able to filter out the back-reflected signals of water layers in the hydrated biofilm and allowed for direct biofilm quantification. The overall depth-resolved scattering intensity of the biofilm showed very strong positive correlation with CV assay quantification (Spearman’s ρ = 0·92) during the growth phase of the biofilm.CP-OCT was able to quantify the mass of the biofilm by measuring the overall depth-resolved scattering of the biofilm.CP-OCT has the ability to nondestructively monitor biofilm growth and elucidate the growth characteristics of these microcosms on different dental material compositions.