The current study was planned to detect some pathogenic microorganisms that may contaminate raw milk beside application of molecular methods in a trail to detect virulence genes in the isolated bacteria. A grand total of 100 samples of raw milk were randomly collected from small breeders and supermarkets then transferred directly to the laboratory with a minimum of delay, where they were examined bacteriologically for detection of some food poisoning bacteria including; Escherichia coli, Staphylococcus aureus and Salmonella. The obtained results clarified that the prevalence of E. coli, Staph. aureus and Salmonella in raw milk samples was 64, 35 and 3%, respectively. Serological identification of E. coli isolates revealed that only 4 isolates of E. coli (6.25%) were belonged to Enteropathogenic E. coli where serological identification revealed the presence of O86, O119: H6, O26: H11 and O125: H21 serotypes. Moreover, multiplex PCR was employed for detection of stx1, stx2 and eaeA virulence genes specific for Enteropathogenic E. coli. Also, it was found that the prevalence of Coagulase positive Staph. aureus based on coagulase test was 60% (21 out of 35 isolates) while it was 54.3% based on presence of clfA gene. Finally, serological identification of Salmonella isolates revealed the presence of S. Infantis, S. Typhimurium and S. Enteritidis. The recorded results in the current work highlighted the role of raw milk in transmitting food poisoning bacteria to consumers so strict hygienic measures must be considered during production and distribution of raw milk.