The current study was performed to investigate the ability of propolis as natural preservative of buffalo milk against contamination by Escherichia coli. Here, 100 milk samples were collected from buffalo and transported in icebox to a laboratory. These samples were subjected to bacterial cultivation and diagnostic tests, to recover E. coli from the milk samples and to test its antibiotic susceptibility profile. Each isolate was subjected to virulence gene- and 16S rRNA gene-based PCRs and sequencing. The propolis, collected from honey bee colonies, was exposed to an extraction process and GC mass analysis. For the preservation experiment, finely-ground propolis was prepared with different concentrations (10mg,20mg, and 40mg). Each concentration was added to 1 ml of milk with a control 1ml of milk with no propolis. After that, all milk groups were placed in 5±1 °C for 10 days. Later, bacterial count was done at zero-time, 3 days, 7 days, and 10 days to identify the bacterial contamination. E. coli was detected in the milk samples before adding propolis as confirmed by the PCR. The bacterium demonstrated high sensitivity to gentamycin (18.66±0.88mm) and amikacin (15.66±0.33mm) with very low sensitivity that reached 0 mm in amoxicillin and tetracycline. The finding of the bacterial counts revealed significant (p<0.05) protection against bacterial growth, especially at high concentrations. The sequencing revealed distinct but close similarity with world isolates of E. coli. The gene expression of the virulence genes was seen significantly (p<0.05) affected by propolis. The study demonstrates that the propolis protects against bacterial growth.