Upfront MRI is taking the lead in the diagnosis of clinically significant prostate cancer, while few image-guided biopsies (IGBs) fail to demonstrate clinically significant prostate cancer. The added value of innovative biomarkers is not confirmed in this context. We analysed SelectMDx-v2 (MDx-2) in a cohort of upfront MRI and image-guided biopsy patients. Participants included patients who received a trans-rectal elastic-fusion registration IGB on the basis of DRE, PSA, PCA3, and PCPT-2.0 risk evaluation. Pre-biopsy MRI DICOM archives were reviewed according to PI-RADS-v2. Post-massage first-void urine samples stored in the institutional registered bio-repository were commercially addressed to MDxHealth to obtain MDx-2 scores. Univariate and multivariate analyses were conducted with the detection on IGB of high-grade (ISUP 2 and higher) as the dependent variable. High-grade cancer was demonstrated in 32/117 (27.4%) patients (8/2010–8/2018). Age, prostate volume, biopsy history, MDx-2, and PI-RADS-v2 scores significantly related to the detection of high-grade cancer. MDx-2 scores and the clinical variables embedded into MDx-2 scores were analysed in multivariate analysis to complement PI-RADS-v2 scores. The two combinations outperformed PI-RADS-v2 alone (AUC-ROC 0.67 vs. 0.73 and 0.80, respectively, p < 0.05) and calibration curves confirmed an adequate prediction. Similar discrimination (C-statistics, p = 0.22) was observed in the prediction of high-grade cancer, thereby questioning the respective inputs and added values of biomarkers and clinical predictors in MDx-2 scores. Based on the results of this study, we can conclude that instruments of prediction developed for systematic prostate biopsies, including those that incorporate innovative biomarkers, must be reassessed and eventually confirmed in the context of upfront MRI and IGB.