Streptococcus is a genus of spherical Gram-positive bacteria belonging to the phylum firmicutes and the lactic acid bacteria group. Members of the genus Streptococcus cause mild to severe bacterial illnesses in humans and animals. This work was designed to determine the biochemical, serological, and molecular characterization of Streptococci species isolated from cow`s milk, broilers chickens, and Nile tilapia. Bacteriological examination was carried out on 255 Samples (120 cow' milk, 65 broiler chickens, and 70 Nile tilapia) collected during the period from July 2010 to June 2013 from various localities in Beni-Suef Governorate, Egypt. Identification of the isolated strain revealed S. agalactiae, S. equi subsp.  zooepidemicus, E. fecalis, S. iniae, E. avium and L. lactis subsp. Lactis were found in the isolated strains. Furthermore S. agalactiae (Lancefield group B), S. equi subsp.  zooepidemicus (Lancefield group C) and E. faecalis (Lancefield group D) were found in the agglutination test for Lancefield groups. A multiplex PCR assay involves amplifying the multiple gene products in a single reaction using primers derived from 16S rRNA genes of S. agalacitae, S. equi subsp. zooepidemicus and lctO gene of S. iniae. The amplified products showed amplification of 220bp, 679bp and 870bp of amplification respectively. The sensitivity of streptococci species to antimicrobials exhibited that S. agalactiae, S equi subsp. zooepidemicus and S. iniae showed high sensitivity to both enrofloxacin, erythromycin followed by cephalexin.