Abstract: Objective: This study was aimed to evaluate the antimicrobial activity of different parts of Sepia officinalis (S. officinalis) which was collected from Alexandria Mediterranean waters, Egypt against the most common human pathogens and detection of the most active part and extract. Materials and Methods: Different parts from Sepia officinalis were screened for antimicrobial activity in vitro. Acetone, chloroform, ethanol, methanol and aqueous extracts were tested against some pathogens, four Gram-negative bacteria: Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa), Salmonella typhi (S. typhi), Vibrio cholera (V. cholera) and two species of clinically human pathogenic Gram positive: Bacillus subtilis (B. subtilis) and Staphylococcus aureus (S. aureus) and two fungal strains such as Aspergillus fumigatus (A. fumigatus) and Candida albicans (C. albicans). Results: Maximum antibacterial activity was noted in methanol extract of ink against P. aeruginosa and S. typhi (18 and 15 mm, respectively), followed by methanol and chloroform extracts of nidamental gland and ink against P. aeruginosa (14 and 13.2 mm, respectively). On the other hand, the minimum antifungal activity was observed against the tested fungi but chloroform and methanol extracts were also the best solvents. No antimicrobial activity was detected in aqueous extracts of different parts. Conclusion: The screening result confirms that ink and nidamental gland extracts had a good antimicrobial activity to play a vital role in future for medical applications.