Nitzschia sp is a marine microalgae that play a crucial role in aquaculture and can be used to enhance the nutritional value of food. Nitzschia sp contain a protein with the potential to be use as a novel antimicrobial agent. This study aimed to isolate and fractionate active protein from microalgae Nitzschia sp. and evaluate its ability to inhibit bacterial growth. Nitzschia sp. was cultivated, then was centrifuged to separate its filtrate from the biomass. Biomass compotition contains 39.98% protein was measured by Kjeldal method. Protein was isolated from the biomass used salting-out method by adding ammonium sulfate at saturation levels of 0-20%, 20-40%, 40-60%, and 60-80%. Protein was purified by a dialysis method using a cellophane membrane. The protein content was determined by the Lowry method. Antimicrobial activity was determined by using disk diffusion method. The results showed that the protein content of each fraction of 0-20%, 20-40%, 40-60%, and 60-80% were 10.3 mg/mL, 0.93 mg/mL, 1.61 mg/mL, and 1.91 mg/mL, respectively. The antibacterial study showed that protein fractions have an inhibitory effect on the growth of pathogenic bacteria Salmonella thypi (fraction 0-20%) with the largest inhibition zone of 14.3 mm and Staphylococcus aureus (fraction 0-20% and 60-80%) with the largest inhibition zone of 14.7 and 8.2 mm, respectively. Protein fractions from Nitzschia sp. were found to be effective against both gram positive Staphylococcus aureus and gram negative Salmonella thypi.