This study focused on the investigation of Actinomycetes strains isolated from sediment sample of Cheliff River estuary in Mostaganem (North-west of Algeria). Identification of the 5 strains of Actinomycetes isolated from sediments by morphological, biochemical methods and by16S rRNA gene sequence analysis revealed that four strains belonged to the genus Streptomyces and one - to the genus Nocardia. No strains of Actinomycetes were isolated from water samples. The optimum growth of all strains was recorded between 28°C and 30°C and at pH 7-9. All strains had halotolerant and mesophile behavior except SSG which grew very well at 45°C and could be considered as thermotolerant bacteria. Antibacterial activity was tested against five pathogenic bacteria as Escherichia coli ATCC 25922, Staphylococcus aureus ATCC 6538, Salmonella typhi ATCC 9289, Bacillus subtilis ATCC 6633 and Pseudomonas aeruginosa ATCC 2785. All isolated strains exhibited an inhibitory activity against at least one human pathogenic bacterium. Streptomyces sp. strain SSG showed antibacterial effect against four pathogenic tested bacteria followed by Streptomyces sp. SSB1, Streptomyces sp. SSM and Nocardia sp. NSR. This study revealed that Cheliff River estuary contains Actinomycete species (Streptomyces and Nocardia) with proved antibacterial activity and could be a new source for isolation of antibiotic producing bacteria against human pathogenic bacteria