The antimicrobial activity of a methanolic extract of Allocasuarina littoralis leaves was investigated by disc diffusion assay against a panel of bacteria and fungi. A. littoralis leaf extract inhibited the growth of 8 of the 14 bacteria tested (57%). Gram-positive and Gram-negative bacteria were both inhibited by A. littoralis extract although Gram-positive bacteria were more susceptible. 5 of the 11 Gram-negative bacteria (45%) and 100% of the Gram-positive bacteria tested had their growth inhibited by A. littoralis extract. A. littoralis leaf extract displayed no antifungal activity towards any of the fungi when tested by disc diffusion assay. The antibacterial activity of A. littoralis leaf extract was further investigated by growth time course assays which showed significant growth inhibition in cultures of Bacillus cereus, Bacillus subtilis and Pseudomonas fluorescens within 1 h but not of Aeromonas hydrophilia.