Background: Mastitis, which refers to as infection of the mammary gland, is considered one of the important wide-ranging diseases that affect dairy animals that lead to decreasing of somatic cell contents (SCC) and milk amount. On the other hand, sub-clinical mastitis does not appear to produce any changes in the udder or milk. However, subclinical mastitis is a severe problem and acts as a pathogen pool that transmits udder infection across animals in the farm. Objective: The aim of the current work was phenotypic and molecular identification of some bacterial species causing animal mastitis in Basrah province, Iraq. Materials and methods: This cross sectional study was conducted in Basrah City from October 2018 to March 2019. A total of 50 milk samples were collected from cows with suspected clinical mastitis cases and transported immediately to laboratory unit and cultured on appropriate culture media like Blood agar, MacConkey agar (MA) and Mannitol salt agar (MSA) respectively. Thereafter, the pure isolate were identified by biochemical tests and molecular tests using 16SrRNA characteristics. Results: The most common causative agents were Staphylococcus aureus that isolated from cases and the other microorganisms were Bacillus spp., Escherichia coli and Enterobacter. Conclusion: S. aureus and E. coli were the most predominant bacteria causing animal mastitis. The 16s rRNA gene was a good tool for identification of bacteria causing mastitis for both Gram positive and Gram-negative bacteria where in concern with the morphological tests.