The use of aromatic and medicinal plants for therapeutic aims has developed since ancient times because of their richness in bioactive substances. Research was carried out to explore the most suitable and standardized method to extract bioactive molecules from plant. The aim was to test the effect of the change of extraction time (24, 48, 72 h) and the use of different solvents (water, ethanol) on both phytochemicals levels and antibacterial activity of Citrullus colocynthis (L.) Schrad. extracts.  The results revealed that the time of extraction and the nature of the solvent used had a considerable influence on the level of the molecules compounds. The variation of the time correlates with the extraction yield because changing extraction time from 24 h until 72 h the extraction yield with ethanol rose. However, extraction yield decreases inversely when the time of extraction increases by water as solvent. The ethanol extract after 72 h extraction was characterized by the highest phenolic content 61.58 mg gallic acid equivalent per g dry weight. Ethanolic extracts 24 h and 72 h and aqueous extract 24 h were the most active against S. aureus with inhibition zone 20, 18 and 16, respectively. The results, also, showed that the aqueous extract after 72 h had no action on all bacteria strains.