Objectives Plants were used as medicines thousands of years ago. Conventional medicine use is increasing and many of the currently used drugs are extracted from herbal sources. In Palestinian traditional medicine, the Alhagi mannifera plant is used for the treatment of cancer. Our study aimed to extract this plant using five solvent fractions, identifying their chemical compositions, and evaluating their antimicrobial and cytotoxic effects. Methods The successive technique was used to extract five solvent fractions of A. mannifera . While the spectral analysis was used to characterize quantitatively and qualitatively the chemical components of these extracts. The antimicrobial activity of plant extracts was evaluated against seven microbial strains using a broth micro-dilution assay. The cytotoxic activity was assessed using a 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay against cervical cancer cell line (HeLa). Results A total of 165 compounds were identified in A. mannifera different extracts. In the petroleum ether extract were found a total of 55 compounds. The major compounds were 2,5-cyclooctadien-1-ol (9.42%), 3-chloropropionic acid, heptyl ester (9.42%), carbonic acid, ethyl nonyl ester (9.42%) and chloroacetic acid. In methylene chloride extract a total of 11 compounds were found, and the major compounds were m-ainobenzenesulfonyl fluoride (14.35%), dodecane,2,6,10-trimethyl- (14.35%) and propanoic acid,2,2-dimethyl-,2-ethylexyl ester (14.35%). In chloroform extract, a total of 23 compounds were found. The major compounds were 5-ethyl-1-nonene (21.28%), and decanedioic acid, bis(2-ethylhexyl) ester (21.28%). In acetone extract were found a total of 47 compounds and the major compound was phenol,2,4-bis(1,1-dimethylethyl)- (5.22%). In methanol extract a total of 29 compounds were found and the major compounds were 3-o-methyl- d -glucose (10.79%), myo-inositol, 2-c-methyl- (10.79%), myo-inositol, 4-c-methyl- (10.79%), and scyllo-inositol,1C-methyl- (10.79%). All extracts showed antimicrobial activity. However, the petroleum ether extract showed the most potent antimicrobial effect against Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia , MRSA, and Candida albicans with minimal inhibitory concentration (MIC) values of 1.25, 1.25, 6.25, 0.325, 6.25, and 1.56 μg/mL, respectively. De facto, chloroform extract followed by ether extract displayed potential cytotoxic activity with IC 50 values of 0.2 and 1.2 mg/mL, respectively. Conclusions A. mannifera was found to contain a variety of phytochemicals and its chloroform extract showed a potent cytotoxic effect on HeLa cancer cells. In addition, petroleum ether showed potent antimicrobial agents and these extracts look promising as drug candidates. Further in vivo investigations should be conducted to provide the basis for developing new cancer and microbial infections treatments.