Microbiological studies were carried out on microbial contamination and antimicrobial activity of sea cucumber Holothuria polii collected from Mediterranean Sea at Abu-kir shore of Alexandria, Egypt. The obtained results revealed the presence of isolates of five human Gram-negative pathogenic bacteria, representing five genera were identified to species level, including, Esherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Salmonella sp. and Shigella sp. In addition, an yeast Candida albicans was isolated. The pathogenic bacteria were identified using API 20E strip system (BioMereux). All collected H. polii specimens were healthy with no external signs of infection. Histopathological study of the tegument, intestine and gonads showed no abnormal changes. The antimicrobial activity of two tegumental ethanol extracts (A and B, differ in the method of dehydration) were tested against wide range of pathogenic bacteria and fungi, including intestinal, skin and nosocomial pathogens and one plant fungal pathogen. The results revealed a remarkable antifungal activity of the extract B at 2.5 mg/ml MIC90, especially on Aspergillus niger, Scloretium sp, C. albicans, Aspergillus flavus and Malassezia furfur, and limited antibacterial activity against Gram-negative bacteria (Salmonella choleraesuis ATCC 14028 and Aeromonas hydrophila). The domain of bacterial and limited fungal contamination confirms the results that showed strong antifungal activity of investigated extract.