Background: Citrullus colocynthis L. (C. colocynthis) is a medicinal plant with a long history of traditional usage in Saudi Arabia, particularly for the treatment of digestive issues such as indigestion and stomach pain.Methods: C. colocynthis seeds were examined using qualitative and quantitative phytochemical methods. Antioxidant testing was conducted using DPPH assay and the reducing power test. Antibacterial properties were evaluated using the well-diffusion test, minimum inhibitory concentration (MIC) test, and minimum bactericidal concentration (MBC) test. The effectiveness of the extract against Aphis craccivora (A. craccivora) was assessed using a leaf-dip bioassay, measuring generation time (GT), net reproduction rate (R0), intrinsic rate of increase (rm), doubling time (DT), and finite rate of growth (λ).Results: The methanolic extract of C. colocynthis seeds included diverse bioactive compounds, a good quantity of phenolic content (60.45 mg GAE/g) and flavonoid content (46.66 mg/g). The antibacterial tests showed that the extract was effective only against Staphylococcus aureus and Proteus mirabilis. The extract recorded inhibition zones of 14.5 ± 0.7 mm and 10.5 ± 0.5 mm for Staphylococcus aureus and Proteus mirabilis, respectively. Staphylococcus aureus showed the greatest sensitivity, with MIC of 6.25 mg/mL and MBC of 25.0 mg/mL. The extract was found to have a fatal concentration (LC50) of 9.02% and a lethal concentration (LC95) of 20.50% against A. craccivora aphid.Conclusions: The current vitro study on C. colocynthis reported that the seeds are effective as antioxidants, antibacterial agents (only against Gram-positive bacteria), and aphicidal agents against aphids. More investigations are recommended to examine the possible toxicity and biochemical interactions in vivo.Keywords: Plants; Biological activity; In vitro study; Citrullus colocynthis; Aphis craccivora; Aphicidal