Purpose: The study was conducted to optimize the extraction process with high polyphenol and flavonoid content from O. gratissimum L. and evaluate their biological activities. Research Method: The extraction of O. gratissimum L. extract was performed by combining ultrasound-assisted and hot extraction methods. Findings: The results showed that extraction of O. gratissimum L. was performed under optimal conditions by response surface methodology (RSM), including ethanol concentration of ۷۰%, raw material: ethanol ratio of ۱: ۳۵.۷۱ (g.g-۱), time extraction of ۶۹.۷۷ min, and at a temperature of ۸۹.۸۲ °C, with a total polyphenol content and a total flavonoid content of ۱۹۰.۹۸۷ (mgGAE.gDW-۱) and ۲۷.۱۰ (mgQE.gDW-۱), respectively. The extract was concentrated by vacuum rotary evaporation to obtain the total extract which was subjected to evaluation for its physicochemical components and biological activities. The results showed that the total extract quality met the current standards of Vietnam without heavy metals and pesticide residues. In addition, the antibacterial, antioxidant and anti-inflammatory properties of the total extract sample from O. gratissimum L. also gave positive results, confirming the significant biological activity. Research limitations: No limitations were encountered. Originality/Value: Ocimum gratissimum L. is a medicinal herb widely used in traditional medicine across East Asia, especially in Vietnam. Due to the high content of physiochemical components, including essential oils and phenolic compounds such as polyphenols and flavonoids, O. gratissimum L. exhibits antioxidant, anti-inflammatory, and antibacterial properties. Therefore, studies on the biological activity of solution extracts and total extracts from O. gratissimum L. are essential for evaluating the potential applications in the fields of medicine, foods, and cosmetics.