Background: Marine seaweeds synthesize different metabolites with various biological activities, including antimicrobial effects. Objectives: The main object of this study was to fractionate the seaweed Padina australis and analyze the antimicrobial activities of these fractions to isolate the bioactive compounds. Methods: Padina australis was collected from the Persian Gulf and extracted by maceration with methanol-ethyl acetate solvent. The extract was evaporated and partitioned by the Kupchan method to yield hexane, dichloromethane, water, and butanol partitions. Dichloromethane partition was further fractionated by MPLC, and antimicrobial activity of the crude extracts and all fractions were investigated using disc diffusion and agar plate count methods. The MIC values of hexane, dichloromethane, chloroform, and butanol partitions and all fractions were calculated. Results: Fraction numbers 11, 4, and 7 showed the highest inhibitory effect with MIC value of 87 µg/mL, 156 µg/mL, and 156 µg/mL against E. coli, P. aeruginosa, and S. aureus, respectively. Conclusions: This study reveals that different fractions of P. australis have antimicrobial activities against various microorganisms.