Necrotic enteritis due to Clostridium perfringens strains harboring the netB gene is a well-known disorder in poultry. The aim of this study was to investigate the association of a novel bacteriocin, perfrin, with netB among isolates from healthy and diseased ostriches and broiler chickens. Forty-six C. perfringens isolates from broiler chickens and ostriches collected from 2010 to 2014 were included in this study and subjected to PCR to detect netB and perfrin genes. Six (60%) and 9 (25%) isolates were positive for both netB and perfrin genes in broilers and ostriches, respectively. Statistical analysis found a significant difference between healthy and diseased flocks for perfrin both in broilers and ostriches. For netB, the significant difference was only found between healthy and diseased ostrich flocks. This is the first report of the presence of perfrin in netB-positive C. perfringens strains in ostriches.