Staphylococcus aureus a gram-positive bacteria cause diseases through direct destruction and invasion of tissue by the production of toxins and Escherichia coli a gram-negative bacteria causes a variety of diseases. Testing of antibacterial susceptibility plays a major role in antibacterial treatment. Their are some factors which affects the results are quality, type and structure of a disks. This study was carried out with the aim to determine the Zone of inhibition of antibiotics against Staphylococcus aureus and Escherichia coli using industrially manufactured sensitivity disc. In positive test against Staphylococcus aureus the zone of inhibition was highest in antibiotic streptomycin (10mm/dm) and lowest in Amoxil (0mm/dm). In negative test against Staphylococcus aureus the zone of inhibition was highest in antibiotic Reflacine (10mm/dm) and lowest in Amplicin (2mm/dm). In positive test against Escherichia coli the zone of inhibition was highest in antibiotic Ciproflox (7mm/dm) and lowest in Amoxil (2mm/dm). There is no effect on antibiotic Norfloxacin, Erythromycin, Levofloxacin and Ampiclox. In negative test against Escherichia coli the zone of inhibition was highest in antibiotic Streptomycin (12mm/dm) and lowest in Tarivid (2mm/dm