Blondo is a by-product of Virgin Coconut Oil (VCO) production that still contains protein and has the potential to be utilized as a source of bioactive peptides. This study aimed to isolate short peptides from VCO blondo and evaluate their antibacterial activity against Staphylococcus aureus and Escherichia coli. Peptide isolation was carried out through blondo preparation, lipid extraction using n-hexane, carbohydrate removal using ethanol, protein isolation using phosphate buffer and ammonium sulfate precipitation, followed by dialysis and enzymatic hydrolysis using bromelain. Protein content was determined using the Bradford method with a UV-Vis spectrophotometer at 595 nm. Antibacterial activity was evaluated using the disc diffusion method against Staphylococcus aureus and Escherichia coli. The results showed that the isolated protein contained 95 ppm. The hydrolyzed short peptides exhibited antibacterial activity against Staphylococcus aureus with an inhibition zone diameter of 19 mm, indicating strong antibacterial activity, while no inhibition zone was observed against Escherichia coli. These findings suggest that short peptides derived from VCO blondo have potential as natural antibacterial agents and are more effective against Gram-positive bacteria than Gram-negative bacteria.