Biofilm producing Pseudomonas aeruginosa is a highly versatile opportunistic food borne pathogen that contaminate food and causing serious problems in man, animals and food processing-manufacturing cycles. This research focused on isolation and Identification of P. aeruginosa from normal raw and mastitic cow’s milk from some regions in Baghdad. Study design including collection and processing of sixty milk samples (thirty samples for each type: normal & mastitic) from regions of Abu-Ghraib, Al-Fudhaliyah and Al-Sadrya (twenty samples from each region: ten samples for each type) during period December (2016) to February (2017), in which they collected and processed according to modified dairy microbiological methodology in veterinary public health laboratory using gold standard Cetirmide-Naldixic acid chrome agar (CNP), Electronic RapID™ ONE (4 hours) biochemical panel micro-tubes strep identification system compendium with reference colors chart and online confirmation microcodes data base software and gold standard double staining technique, Microtiter Plate Assay for biofilm formation with methylene blue and safranin dyes. Antibiotics Susceptibility Pattern by Kirby-Bauer technique or disk diffusion method was done according to instructions of clinical laboratory standards institute (CLSI) or national committee for clinical laboratory standards (NCCLS) by using a Muller-Hinton agar and McFarland opacity tubes for checking resistance profile of isolates. Data were analyzed for significant differences by statistical package for social sciences software (IBM SPSS) in which a Chi-square was used. The results revealed isolation and identification of nine strains of P. aeruginosa out of sixty samples (15%): three strains from Abu-Ghraib (5%): two from mastitic (3.33%) and one from normal raw milk (1.66%), two strain from Al-Fudhaliyah (3.33%): one from mastitic (1.66%) and one from normal raw milk (1.66%), and four strains from Al-Sadrya (6.66%): three from mastitic (5%) and one from normal raw milk (1.66%). In conclusion: data revealed contamination of raw milk and infection of dairy mammary glands with P. aeruginosa from some regions in Baghdad, thus we recommend monitoring of milk producing animals and their environment with milk production, transportation and storage stages through application of good management practices and hazard analysis critical control points (HACCP) strategies to overcome or reduce these public health problems.