Background: and Purpose: The rationale for performing a second brain biopsy after initial negativity is not well evaluated in the literature. This study was designed to 1) assess the efficacy of a second brain biopsy when the first biopsy was nondiagnostic, 2) identify possible factors associated with an increased diagnostic rate in the second biopsy, and 3) analyze additional morbidity induced by the second biopsy. Methods: We performed a retrospective cohort study from 2009 to 2019, during which 1,919 patients underwent a brain biopsy, including 30 who were biopsied twice (1.6%). The specific histological diagnosis rate, diagnosis-associated factors, and complication rate were assessed for the 30 twice-biopsied patients. Results: The second biopsy allowed a specific histological diagnosis in 86.7% of the patients who had initially undergone a nondiagnostic brain biopsy [odds ratio (OR)=7.5, 95% confidence interval (CI)=3.0–18.7, p<0.001]. The multivariate analysis showed that only prebiopsy corticosteroid administration (OR=2.6, 95% CI=1.1–6.0, p=0.01) was an important factor in predicting a nondiagnostic biopsy. None of the patients developed a symptomatic complication after the first biopsy, while two (6.0%) patients experienced a transient complication after the second biopsy (p=0.49). Conclusions Performing a second brain biopsy in patients who have an initial nondiagnostic biopsy is effective in most cases. We advocate that a second biopsy be systematically considered in the diagnosis algorithm of these patients after it has been verified that molecular testing cannot help to obtain a diagnosis. Corticosteroid administration can lead to nondiagnostic biopsies and should be avoided when possible during the prebiopsy period.