Our study explored frozen section reliability in prostate cancer (PCa) diagnoses and described surgical steps of a 3D magnetic resonance imaging (MRI)–ultrasound (US)-guided prostate biopsy (PB) and focal cryoablation of the index lesion (IL) in a single-setting procedure. Patients with a suspicious prostatic specific antigen (PSA) value, with a PIRADS 4 or 5 single lesion, were enrolled for trans perineal 3D MRI–US-guided PB and TRUS-guided focal cryoablation. Three cores were taken from the IL, three cores from the surrounding area, while systematic sampling was performed for the rest of the gland. After confirmation of PCa in frozen sections, focal cryoablation was performed. The 1st-year follow-up schedule included a PSA test at a 3-month interval, MRI 3 months and 1 year postoperatively and PB of the treated area at 1 year. Following the follow-up schedule, an involved PSA test at a 3-month interval and yearly MRI were performed. The PCa diagnosis was histologically confirmed in all three patients with frozen sections. At final histology, a single Gleason score upgrade from 6 (3 + 3) to 7 (3 + 4) was observed. All patients were discharged on postoperative day 1. At the 3-month evaluation, mean PSA values decreased from 12.54 (baseline) to 1.73 ng/mL and MRI images showed complete ablation of the IL in all patients. Urinary continence and potency were preserved in all patients. At the 1-year follow-up, one patient had suspicious ipsilateral recurrence on MRI and underwent a new analogous procedure. Post follow-up was uneventful and PSA remained stable in all patients. Three-dimensional MRI–US-guided frozen sectioning and focal cryoablation of the IL is a step forward towards a “patient-tailored” minimally invasive approach to the diagnosis and cure of PCa.