Background: Polymicrobial brain abscess (PBA) is a complex infection caused by two or more pathogens and a life-threatening condition with diagnostic and therapeutic challenges. Methods: We retrospectively identified PBAs in 31 patients (a median of 58 years; 24 male, and 7 female) then examined their clinical, radiological and pathological characteristics. These characteristics of PBAs were compared with those of monomicrobial BAs (MBAs) in the previously reported 113 patients. Results: PBAs and MBAs had a few similarities such as nonspecific clinical presentations and overall similar prognosis following surgical intervention with broad-spectrum antimicrobial therapy. However, PBAs were highly heterogeneous with more complexity on MRI/CT imaging and histopathology. While PBAs were typically rim-enhancing lesions at late-stages, 30/31 (97%) of PBAs showed the lobulation of enhancing rims/walls; on MRI, 14/26 (54%) of cases demonstrated marked difference in the thickness of enhancing rim, marked difference in the degree of DWI signal, and/or marked variation in intra-lesional MRI signal. PBA histopathology was characterized by alternating early-stage and late-stage features with a combinations of 2-4 pathogens. Conclusions: BPAs share some features with MBAs, but they are more heterogeneous with greater complexity on imaging and histopathology. Their diagnosis and disease staging require a clinico-radiologico-pathological approach.