This study's purposes were to determine the yield of repeat direct in-bore magnetic resonance-guided prostate biopsy (MRGB) (MRGB-2) after the first one was found to be negative (MRGB-1), to correlate with clinical parameters, and to present the subgroup analyses of patients with positive repeat biopsies, despite having a negative initial biopsies. We retrospectively included patients with MRGB-2 after a negative MRGB-1 both between January 2006 and August 2016. This study included 62 patients (median age, 63 years; interquartile range [IQR], 58-66 years) with 75 sampled lesions during MRGB-2 left for analysis, and 63 lesions were resampled and 12 new lesions were sampled. Included patients had a prostate specific antigen (PSA) at MRGB-1 of 13 ng/mL (IQR, 5.8-20.0) and a PSA at MRGB-2 of 15 ng/mL (IQR, 9.0-22.5). All anonymized magnetic resonance imaging (MRI) data were retrospectively reassessed according to Prostate Imaging-Reporting and Data System version 2 by two radiologists. Images of MRGB were compared to determine whether the same prostate lesion was biopsied during MRGB-1 and MRGB-2. Descriptive statistics were utilized to determine the yield of clinically significant prostate cancer (csPCa) at MRGB-2. Gleason score of ≥ 3 + 4 was considered csPCa. In 16/75 (21%) lesions csPCa was detected during MRGB-2. Of 63 resampled lesions, 13 (21%) harbored csPCa at MRGB-2. In two patients, csPCa was detected on repeat biopsy, while the volume of the lesion decreased between MRGB-1 and MRGB-2. Patients could benefit from repeat biopsy after negative initial MRGB, especially in the case of increasing PSA values and persisting PCa suspicion in MRI. Further research is needed to establish predictors for positive repeat targeted biopsies.