Materials derived from extracellular matrices (ECMs) are being evaluated as scaffolds for surgical reconstruction of damaged or missing tissues. It is important to understand the susceptibility of these biological materials to bacterial infections. ECMs derived from porcine small intestinal submucosa (SIS) and urinary bladder submucosa (UBS) were found to possess antimicrobial activity. ECM extracts, obtained by digesting these acellular matrices in acetic acid, demonstrated antibacterial activity against Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. Antimicrobial activity was determined using a minimal inhibitory concentration assay. Bacteriostatic activity was detected at protein concentrations of ECM extracts equivalent to 0.77-1.60 mg/mL. ECM extracts were found to inhibit bacterial growth for up to at least 13 h. The resulting extracts consisted of water-soluble peptides and proteins with molecular weights ranging from <4 to >100 kDa and lower molecular weight compounds, as determined by size exclusion liquid chromatography.