Natural constituents have been utilized to avoid humanity from various diseases, such as microbial infection and cancer, over several decades due to bioactive compounds.Myoporum serratumseeds extract (MSSE) was formulated via HPLC for flavonoid and phenolic analysis. Moreover, antimicrobial via well diffusion method, antioxidant via 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging method, anticancer activities against HepG-2 cells (human hepatocellular cancer cell line), and MCF-7 cells (human breast cancer cell line), and molecular docking of the main detected flavonoid and phenolic compounds with the cancer cells were performed. The phenolic acids, including cinnamic acid (12.75 µg/mL), salicylic acid (7.14 µg/mL), and ferulic (0.97 µg/mL), while luteolin represents the main detected flavonoid with a concentration of 10.74 µg/mL, followed by apegenin 8.87 µg/mL were identified in MSSE.Staphylococcus aureus,Bacillus subtilis,Proteus vulgaris, andCandida albicanswere inhibited by MSSE with 24.33, 26.33, 20.67, and 18.33 mm of inhibition zone, respectively. MSSE exhibited a low inhibition zone of 12.67 mm againstEscherichia coliwhile showing no inhibitory activity againstAspergillus fumigatus. The values of MIC ranged from 26.58 to 136.33 µg/mL for all tested microorganisms. MBC/MIC index and cidal properties were attributed to MSSE for all tested microorganisms exceptE. coli. MSSE demonstrated anti-biofilm 81.25 and 50.45% ofS. aureusandE. coli, respectively. IC50of the antioxidant activity of MSSE was 120.11 µg/mL. HepG-2 and MCF-7 cell proliferation were inhibited with IC50140.77 ± 3.86 µg/mL and 184.04 µg/mL, respectively. Via Molecular docking study, luteolin and cinnamic acid have inhibitory action against HepG-2 and MCF-7 cells, supporting the tremendous anticancer of MSSE.