The Gleason grading system is a fundamental indicator of the aggressive nature of prostate cancer (PCa). Diffusion-weighted imaging (DWI) and magnetic resonance (MR) spectroscopy (MRS) are methods for the assessment of PCa aggressiveness. The present study was designed to prospectively investigate whether transrectal ultrasound (TRUS)-guided MR imaging (MRI)-directed biopsies (TRUS‑MR‑Dbs) improve the prediction of PCa aggressiveness in comparison with 12-core TRUS-guided biopsies (TRUS‑Gbs). A total of 518 patients underwent pre-biopsy multi-parametric MRI to identify the clinically suspicious PCa regions. TRUS‑MR‑Dbs were performed on patients with suspected PCa by MRI in addition to TRUS‑Gbs. Only patients who underwent radical prostatectomy (RP) were included in the comparative analysis. TRUS‑biopsy was directed to those areas within suspicious regions with a minimum apparent diffusion coefficient obtained by DWI or with a maximum (choline + creatine)/citrate ratio obtained by MRS. The highest Gleason grades (HGGs) and the Gleason scores (GSs) of specimens were identified. The biopsies and RP results were evaluated using a McNemar test or χ2 analyses using Fisher' exact tests. MRI results were positive in 254 (49.0%) of the 518 patients. TRUS‑MR‑Db detected 165/254 (65.0%) cancer cases and TRUS‑Gb detected 190/518 (36.7%) cancer cases. Forty patients underwent RP. The TRUS‑MR‑Dbs method demonstrated a higher concordance rate (CR) with RP (89.6%) than TRUS‑Gbs (72.9%, P=0.008) for the overall HGG. The CRs with RP for TRUS‑MR‑Dbs vs. those for TRUS‑Gbs were 100 vs. 85.7% (P=0.5), 87.5 vs. 68.8% (P=0.031) and 50 vs. 50% (P=1) for HGG3, HGG4 and HGG5, respectively. The HGG CRs with RP for DWI‑directed biopsies (DWI-Dbs) vs. MRS-directed biopsies (MRS-Dbs) were 77.1 vs. 50.0% (P=0.015) for the overall tumors, 80.0 vs. 40.0% (P=0.003) for peripheral zone tumors and 69.2 vs. 76.9% (P=1) for transition zone tumors. A total of 37 (77.1%) and 25 (52.1%; P=0.007) tumors were assigned accurate GS for TRUS‑MR‑Dbs and TRUS‑Gbs, respectively. The results revealed that TRUS‑MR‑Dbs improved the prediction of PCa aggressiveness and that DWI-Dbs had a superior performance when compared with MRS‑Dbs in the peripheral zone.