Published by John Wiley & Sons Ltd Aim: To explore a set of inflammatory biomarkers obtained from dentinal fluid (DF) from patients with symptomatic irreversible pulpitis (IP), reversible pulpitis (RP) and normal pulp (NP). Methodology: A cross-sectional exploratory study was performed, recruiting 64 patients on the basis of their respective pulp condition. DF samples were obtained from all patients (23, from IP patients; 20, from RP patients; and 21, from NP patients). Quantification of biomarkers was performed using a Luminex® MAGPIX platform system and multiplex assay kits. The Kruskal–Wallis test was used for comparisons with regard to pulp state. A simple logistic regression model and the odds ratio (OR) with a 95% level of confidence (P = 0.05) were used to evaluate associations between biomarker levels and pulpal diagnosis. The performance discrimination of the biomarkers was evaluated through the construction of a receiver operating characteristic (ROC) curve by calculating the area under the curve (AUC) for IP versus RP after logistic regression modelling. Youden criteria were used to establish cut-off points for biomarkers alone with AUC > 70 and P-value < 0.05, or estimated probabilities from the multivariable logistic model. Results: The biomarkers that had significantly higher values in participants with IP versus RP were IL-1α, VEGF-α and FGF acid (P < 0.05). FGF acid (OR: 12.62; P = 0.0085; CI 95% 1.91–83.29) and VEGF-α (OR: 2.61; P = 0.0252; CI 95% 1.13–6.03) were associated with pulp diagnoses of IP versus RP. The AUC-ROC curve for FGF acid was 0.79. The model containing FGF acid, IL-1α, IL-6 and TIMP-1 had an AUC-ROC of 0.92 for IP versus RP with a significant difference from the FGF acid ROC curve (P = 0.0231). Conclusions: Dentinal fluid could be used to assay pulpal mediators in the molecular diagnosis of pulpitis. Despite the limitation of the clinical diagnostics used in the present study, it was possible to detect a difference between irreversible symptomatic pulpitis and reversible pulpitis associated with the following combined biomarkers: FGF acid + IL-6 + IL-1α, +TIMP-1.