| Summary: | Natural origin molecules represent reliable and excellent sources to overcome some medicinal problems. The study of anticancer, anticoagulant, and antimicrobial activities of <i>Thevetia peruviana</i> latex were the aim of the current research. An investigation using high-performance liquid chromatography (HPLC) revealed that the major content of the flavonoids are rutin (11.45 µg/mL), quersestin (7.15 µg/mL), naringin (5.25 µg/mL), and hisperdin (6.07 µg/mL), while phenolic had chlorogenic (12.39 µg/mL), syringenic (7.45 µg/mL), and ferulic (5.07 µg/mL) acids in latex of <i>T. peruviana.</i> Via 1,1-diphenyl-2- picrylhydrazyl (DPPH) radical scavenging, the experiment demonstrated that latex had a potent antioxidant activity with the IC<sub>50</sub> 43.9 µg/mL for scavenging DPPH. Hemolysis inhibition was 58.5% at 1000 µg/mL of latex compared with 91.0% at 200 µg/mL of indomethacin as positive control. Negligible anticoagulant properties of latex were reported where the recorded time was 11.9 s of prothrombin time (PT) and 29.2 s of the activated partial thromboplastin time (APTT) at 25 µg/mL, compared with the same concentration of heparin (PT 94.6 s and APPT 117.7 s). The anticancer potential of latex was recorded against PC-3 (97.11% toxicity) and MCF-7 (96.23% toxicity) at 1000 μg/mL with IC<sub>50</sub> 48.26 μg/mL and 40.31 µg/mL, respectively. Disc diffusion assessment for antimicrobial activity recorded that the most sensitive tested microorganisms to latex were <i>Bacillus subtilis</i> followed by <i>Escherichia coli</i>, with an inhibition zone (IZ) of 31 mm with minimum inhibitory concentration (MIC) (10.2 μg/mL) and 30 mm (MIC, 12.51 μg/mL), respectively. Moreover, <i>Candida albicans</i> was sensitive (IZ, 28 mm) to latex, unlike black fungus (<i>Mucor circinelloides</i>). TEM examination exhibited ultrastructure changes in cell walls and cell membranes of <i>Staphylococcus aureus</i> and <i>Pseudomonas aeruginosa</i> treated with latex. Energy scores of the molecular docking of chlorogenic acid with <i>E. coli</i> DNA (7C7N), and Rutin with human prostate-specific antigen (3QUM) and breast cancer-associated protein (1JNX), result in excellent harmony with the experimental results. The outcome of research recommended that the latex is rich in constituents and considered a promising source that contributes to fighting cancer and pathogenic microorganisms.
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