Nowadays, most of people would passion to using and purchase organic and natural foods without any artificial ingredients. Moreover, mustard is used in producing medicines, pickles, source of edible oils since ancient times, seasoning of meats and chicken. So the present study was undertaken to determine the chemical components of mustard powder, and then used it in grilled beef as a seasoning to improve sensory evaluation, and physicochemical properties, as well as inhibited Escherichia coli, Staphylococcus aureus and increased shelf life of meat. The results indicated that added mustard powder improving sensory evaluation of the grilled meat slices significantly, especially in odour, in the acceptability of meat treated with 2% level of mustard powder. Meanwhile, addition of mustard powder to fresh meat reduced the natural flora to undetectable levels at 4° or 30°C till 2 days from storage. Application of mustard powder was able to reduce E.coli numbers to uncountable levels at zero time during keeping samples.In view of pH, TBA and TVN values, all of the grilled meat slices with added mustard powder decreased by increasing level on mustard powder concentration during storage times. Mustard powder at 2% level has antibacterial, anti-microflora, natural preserve spices in food, improved taste, smell and physical properties of meat and increased shelf life of meat. Finally mustard powder is acceptable to use as spice and natural ingredient to reduce antimicrobial loading in foodstuff. Further study in this area may be helpful for finding of new principle compound.