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Background: The diagnostic process for prostate cancer after a negative biopsy is challenging. This study compares the diagnostic accuracy of micro-ultrasound (mUS) with multiparametric magnetic resonance imaging (mpMRI) for such cases. Methods: A retrospective cohort study was performed, targeting men with previous negative biopsies and using mUS and mpMRI to detect prostate cancer and clinically significant prostate cancer (csPCa). Results: In our cohort of 1397 men, 304 had a history of negative biopsies. mUS was more sensitive than mpMRI, with better predictive value for negative results. Importantly, mUS was significantly associated with csPCa detection (adjusted odds ratio [aOR]: 6.58; 95% confidence interval [CI]: 1.15–37.8; p = 0.035). Conclusions: mUS may be preferable for diagnosing prostate cancer in previously biopsy-negative patients. However, the retrospective design of this study at a single institution suggests that further research across multiple centers is warranted.