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The synthesis of Cyanobacteria‐mediated nanoparticles has attracted great attention due to its ecofriendly, cost‐effective and biocompatible properties. Silver nanoparticles (AgNPs) are used in different areas such as medicine, household products, disinfectants or food storage. The present study aimed the biosynthesis of AgNPs from Cyanobacterial Chroococcus sp. and their antimicrobial activities on some important pathogen bacterial species. According to the obtained data, optimal conditions for synthesis of AgNPs were extract rate 3 : 1 v/v (cell extract/AgNO3), silver salt concentration 10 mM, pH 7, temperature 60 °C and time 30 minutes. The surface plasmon resonance peak of AgNPs was observed at 420–430 nm, and AgNPs were well‐dispersed spherical with an average size of 11–13 nm in diameter by TEM. SEM‐EDX analysis revealed a strong signal of silver. With FTIR analyzes, shift in the absorption bands after the AgNPs formation was confirmed. X‐ray diffraction analysis exhibited that the AgNPs were cubic crystalline shape. Zeta potential of AgNPs showed ‐ 20 mV. The AgNPs strong showed antibacterial activity on an opportunistic bacterial pathogens of Micococcus luteus, Bacillus subtilis and Pseudomonas aeroginosa. The effective and rapid synthesis of Chroococcus‐mediated AgNPs and their strong antimicrobial effects can create an important potential for their use in biomedical and environmental applications. [ABSTRACT FROM AUTHOR]