Background: The presence of medicines in the environment is considered as a serious threat to the human health. The entrance of these substances into the water sources causes soil pollution, which eventually leads to the environmental pollution and it creates some problems for the public health. Also, increasing antibiotic resistant bacteria has attracted the attention of researchers to the use of natural resources such as marine products, for producing new antibiotics. The aim of this study was to evaluate antimicrobial activities of powdered cuttlebone against Klebsiella oxytoca, Staphylococcus aureus, and Aspergillus flavus. Methods: At first, cuttlebones were washed, dried, and powdered. Then, the powdered cuttlebone was characterized. In the next step, its antimicrobial activities were evaluated using agar well diffusion technique, and minimum inhibitory concentration (MIC) was calculated. Results: The powdered cuttlebone was found to be effective against K. oxytoca (24 mm, MIC: 10-1 mg/mL), but no antimicrobial response was found against S. aureus. Also, the powdered cuttlebone antifungal activity and MIC against A. flavus were recorded 23 mm and 10-1 mg/mL, respectively. Conclusion: The obtained results suggest antimicrobial activities of powdered cuttlebone, which are concentration dependent. Furthermore, cuttlebone can be used as an accessible natural source to provide novel, low cost, and safe antimicrobial agents.