Mango seed kernels (MSKs) have been reported to show antioxidant, antibacterial, and anti-inflammatory properties. This study explores the influence of different optimized extraction systems on the extraction of MSK. The effects on gallic acid (GA) content, total phenolic content (TPC), total flavonoid content (TFC), antioxidant, antimicrobial, and hemolytic activity of MSK extracts from different extraction systems (65.45% ethanol-ultrasound assisted extraction [UAE], 62% ethanol-incubator shaker, 19.4% ethanol-UAE, and 100% water-UAE) were assessed. Based on the results, a nonsignificant difference in phenolic (p = 0.222), flavonoids (p = 0.058), antioxidant (p = 0.165), and antimicrobial activity (p = 0.193) against Staphylococcus aureus whereas a significant difference (p < 0.0001) in hemolytic, GA content, and antimicrobial activity against Clostridium perfringens was observed. Among different extraction systems, aqueous extraction showed significantly lower hemolytic (1.09%) and higher GA content (4.72 mg/g) and comparable results in all other experiments; yield (32.40%), TPC (58.79 mg/g), TFC (2.16 mg/g), and antioxidant (73.19%). Hence, it has been concluded that aqueous extraction system could be considered a sustainable extraction system for practical applications.