The clinical impact of the laterality of perineural invasion (PNI) by prostate cancer remains poorly understood. We herein compared radical prostatectomy (RP) findings and long-term oncologic outcomes in patients with prostate cancer with PNI in two prostate biopsy (PBx) sites. We retrospectively assessed 170 consecutive patients undergoing systematic sextant PBx where PNI had been detected in two of six PBx sites, followed by RP. PNI occurred unilaterally in 140 (82.4%) cases and bilaterally in 30 (17.6%) cases. Compared with unilateral PNI, bilateral PNI was significantly associated with a higher number of cancer-positive sites and longer total tumor length on PBx. However, there were no significant differences in RP findings, including tumor grade/stage and tumor volume, between unilateral and bilateral PNI cohorts. Kaplan-Meier analysis revealed that patients with bilateral PNI had a significantly higher risk of disease progression after RP than those with unilateral PNI (P = .038). In multivariate analysis, bilateral PNI (vs unilateral PNI) showed significance for progression (hazard ratio, 2.281; P = .023). In PBx specimens exhibiting PNI in two sextant sites, bilateral PNI was found to be associated with poorer prognosis as an independent predictor but not worse histopathologic features in RP specimens compared with unilateral PNI.