Nyctanthes arbor-tristis Lin (family Oleaceae), which has been used traditionally for various medicinal purposes in the Indian subcontinent since long before. In view of this, the present study was designed to investigate for chemical constituents, anti-microbial and cytotoxic by methanolic extract of Nyctanthes arbor-tristis. Phytochemical screening revealed the presence of alkaloid, flavanoid, reducing sugar and steroid. In antimicrobial assay, the methanolic extract of the experimental plant was screened to antimicrobial activity against a wide range of both gram-positive and gram negative bacteria by disc diffusion method. The results obtained were compared with that of standard antibiotic, ciprofloxacin. In the brine shrimp lethality bioassay, the LC50 values of the sample was (0.11) μl/ml where the LC50 values of the standard potassium dichromate was (124.13) μl/ml as a positive control. The methanolic extracts are promising and further bioactivity-guided investigation can be done to find out potent antitumor and pesticidal compounds. This study shows that the methanolic extract of Nyctanthes arbor-tristis leaves has antimicrobial and cytotoxic activity