Hemi-ablation of prostate cancer (PCa) requires an accurate prediction of laterality. Recently, multi-parametric magnetic resonance imaging (mpMRI) has recently been increasingly used to enhance clinical staging and characterization of tumor foci. Thus, we tried to investigate the real-life accuracy of combinatory approach of using both transrectal ultrasound (TRUS)-guided prostatic biopsy and mpMRI in predicting the laterality of PCa. We reviewed the records of 335 men who were suspected of having unilateral PCa on multi ( 12)-core TRUS-guided biopsy and preoperative mpMRI and subsequently had undergone radical prostatectomy (RP) at our institution. Based on the analysis of pathologic outcomes from RP, the performance of the combinatory approach in predicting the pathological laterality of PCa was evaluated. Pathology was classified to be unfavorable when showing a Gleason pattern of 4/5 or pT3/N1 features. Significant cancer was defined as non-organ-confined disease, having a Gleason pattern of 4/5, or showing a cancer volume of 0.5 mL. Among the 335 unilateral lobes not suspected to harbor tumor from either the TRUS biopsy or mpMRI, the actual absence rate of malignancy was only 13.7% from a pathologic analysis of RP specimens. Even among the 115 D’Amico low-risk group, the absence rate of malignancy was only 26.1% in unilateral lobes not suspected to harbor tumor. Among the 335 lobes, unfavorable pathology and significant cancer were not observed in 36.1% and 30.7%, respectively. The absence rates of unfavorable pathology and significant cancer among the D’Amico low risk group were 56.5% and 47.8%, respectively. Meanwhile, the absence rate of dominant Gleason pattern 4 or 5 was 74.9% among the 335 total subjects. Our real-life clinical experience showed that the combination of multi-core TRUS-guided biopsy and mpMRI did not provide reliable accuracy in the prediction of true unilaterality of PCa.