Staphylococcus aureus (S. aureus) is the almost-universal cause of furuncles, carbuncles, and skin abscesses which is the most commonly identified agent responsible for skin infections. Most strains of S. aureus are now resistant to antibiotics, forcing the patients treated with high dosage drug, of which must be observed closely for potentially severe adverse effects. Efforts, therefore, have been increasingly made and paid attention to antimicrobials derived from the plants either as pure compounds or as standardized plant extracts. Among a variety of natural sources, seeds from citrus plant are reported to have antioxidant, anti-mutagenic, anti-inflammatory and antibacterial properties. Accordingly, successful isolation and extraction of phytochemicals from lemon seed would be medicinally advantageous. In this study, the antimicrobial efficacy of lemon seed extract against the growth and development of S. aureus was thoroughly investigated. The mixture of lemon seed extract with different concentrations and ratios, and a certain amount of medium and inoculum, of which ranging from 5% to over 20% to determine the Minimum Inhibitory Concentration (MIC) against S. aureus. The obtained results of this study have indicated that the extract of lemon seed showed not only the statistically significant reduction of S. aureus colony formation at 15% in agar test and 22% at broth test with the ratio of 1:4 v/v but also the improved medicinal advantages of aqueous extracts of lemon seed usage against the growth and development of S. aureus. In summary, the lemon seed extract could effectively inhibit the growth of S. aureus at the selectively applied concentration.