In the present study, one hundred samples of raw cow milk were collected randomly from different dairy shops and markets in Zagazig city for isolation and identification of Escherichia coli which is considered a reliable indicator for fecal contamination and an important cause of food poisoning. Identification was done microscopically, biochemically by different biochemical tests (IMVIC) and serologically. The incidence of E. coli in raw milk samples was 47%. Also, the serological identification of E. coli isolates revealed that O26 is the most predominant serogroup by percentage of 21.3%. E. coli pose the greatest threat to human health because of its growing resistance to antibiotics. Antimicrobial susceptibility testing (AST) was done by disc diffusion method against 10 antimicrobials and the results revealed that E. coli isolates were highly resistant to amoxicillinclavulanate, ampicillin, cefotaxime and ceftazidime with percentages of 89.4%, 89.4%, 100.0% and 100.0%, respectively. However, they were highly sensitive to chloramphenicol, ciprofloxacin and tetracycline with percentage of 100.0%, 100.0% and 93.6% respectively. In addition, 89.4% of E. coli isolates showed multi drug resistance (MDR). The ability of bacteria for adherence to food surfaces and biofilm formation is a source of food contamination that affect food safety and industry. Micro titer plate assay used for testing biofilm formation and represented that 78.7% of E. coli isolates were non-biofilm producers, 6.4% were weak biofilm producers, 14.9% were moderate biofilm producers and none of isolates was strong biofilm producers.