Bacteria are among the most dangerous types of pathogens to humans because they are single-celled organisms that cannot be seen with the naked eye. They also attack various body systems and cause many bacterial infections and diseases of the digestive and respiratory systems, among others. Bacteria are regularly finding new behaviors to attack antibiotics, and these ways are spreading. For centuries, plants have been a chief in medicinal handlings for a variety of ailments due to their ability to synthesize hundreds of chemical compounds that play a role in defense against bacteria, fungi, and insects. Lemon (Citrus aurantifolia) is an important plant that typically grows in tropical climates. In this work, the antibacterial activity of citrus juice was evaluated on two nominated pathogenic bacteria, Staphylococcus aureus and Escherichia coli. 100% concentrated and 50% diluted fresh lemon juice tested and was compared with two types of antibiotics Amikacin and Piperacillin. To assay the antibacterial properties, agar well diffusion was followed. The results showed that concentrated lemon juice has an antibacterial effect on both Staphylococcus aureus and Escherichia coli, with the highest inhibition zone reaching 19 mm in Staphylococcus aureus, while the 50% diluted juice had no effect on Escherichia coli. The antibiotics used in this work did not appear inhibition zone in any species of bacteria which were used in this study. This indicates that citrus juice has antibacterial activity against bacteria that have drug resistance to some types of antibiotics because it has actual chemical compounds to eradicate bacteria