The antimicrobial activity of the ethyl acetate crude extract from the sea star Pentaceraster cumingi, collected from Ras Ghareb, the Red Sea, Egypt, was assessed against Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus faecalis, and Candida albicans (used as a fungal model). The ethyl acetate extract exhibited broad antimicrobial activity against all tested bacteria, showing stronger effects against Gram-positive bacteria. The largest inhibition zone of 25.3 ± 0.47mm was observed for E. faecalis. The minimum inhibitory concentration (MIC) was 8mg/ mL for E. coli, P. aeruginosa, and C. albicans, while it was lower (4mg/ mL) for S. aureus and E. faecalis. The minimum bactericidal concentration (MBC) to MIC ratio for all tested strains was ≤ 4, indicating that the crude extract of P. cumingi has both bactericidal and fungicidal properties. GC-MS analysis revealed various bioactive components in the extract. The main compounds identified were hexadecanoic acid (palmitic acid), tetradecanoic acid, palmitoleic acid, pentadecanoic acid, oleic acid, and 9-octadecenoic acid (Z)-methyl ester. These compounds are fatty acids, which have been shown in previous studies to possess strong antimicrobial effects. Molecular docking analysis of these compounds with DNA gyrase B of P. aeruginosa (a model for Gram-negative bacteria) and E. faecalis (a model for gram-positive bacteria) showed that oleic acid was the top binder, with a ΔG of -5.92 kcal/mol for P. aeruginosa and -6.39kcal/ mol for E. faecalis. For C. albicans, tetradecanoic acid demonstrated the strongest binding to the ERG2 protein, with a remarkably low ΔG of -8.1616kcal/ mol. These results indicate that the crude extract of P. cumingi exhibits significant antimicrobial properties, suggesting its potential use in various applications, including wastewater disinfection, biofouling reduction, and pharmaceuticals.