Background Periprostatic infiltration anesthesia (PPIA) and intrarectal topical anesthesia (IRTA) are recommended methods to control pain in transrectal ultrasonographic prostate biopsy (TRUS-Bx). This study evaluates the factors affecting pain during TRUS-Bx, considering the pathologies involved in anorectal pain etiology and comparing the effectiveness of local anesthesia techniques in providing patient comfort. Material and Methods We retrospectively evaluated 477 consecutive patients with TRUS-Bx for elevated Prostate Specific Antigen (PSA), abnormal rectal examination findings, or both. Patients were grouped as local anesthesia methods for pain control during TRUS-Bx. Both groups were compared in terms of age, body mass index, clinical T stage, PSA, prostate volume, number of biopsy cores, type of anesthesia, previous biopsy history, and presence of prostate cancer. We used a visual analog pain scale (VAS) to evaluate the patient's pain status; pre-procedure (VAS-0), during probe insertion (VAS-I), administration of anesthetic (VAS-A), and simultaneous with the biopsy procedure itself (VAS-Bx). For PPIA and IRTA, 4 ml lidocaine 20 mg/ml injection and 5 g 5% prilocaine-5% lidocaine cream was used, respectively. Results The PPIA was used 74.2% (n=354) and IRTA was used for 25.8% (n=123) patients. VAS-0, VAS-I, and VAS-A scores are similar between groups. VAS-Bx was significantly higher in the IRTA than in the PPIA (3.37 ±0.18 vs. 2.36 ±0.12 p0.001). Clinical T stage (OR: 0.59), number of biopsy cores (OR: 1.80), and type of anesthesia application (OR: 2.65) were independent variables on TRUS-Bx for pain. Conclusion Three factors play roles as independent variables associated with the pain in TRUS-Bx; abnormal rectal examination findings, collection of more than twelve core samples during the biopsy, and the type of anesthesia used. Compared with PPIA, IRTA does not improve pain related to probe insertion, and using IRTA results in higher pain scores for biopsy-related pain. Thus, we recommend a PPIA to lower biopsy-related pain.