Ocimum plants are of great interest to traditional medicine in the history of several civilizations, particularly in terms of chronic human diseases. Essential oils obtained from this genus have also been used as therapeutic agents. In this present work, two plants of the Ocimum species from Djibouti, Ocimum basilicum L. and Ocimum americanum L., were subjected to hydrodistillation to obtain their essential oils. Gas chromatography-mass spectrometry was performed to determine the chemical composition of both essential oils. Linalool (41.2%) and estragole (30.1%) are the major compounds among the 37 compounds that have been identified in the essential oil of Ocimum basilicum L. (EOOB), and carvotanacetol (38.4%) and estragole (27.5%) are the main compounds among the 42 compounds that have been identified in the essential oil of Ocimum americanum L. (EOOA). Morever, the cytotoxic activity of EOs was evaluated against 13 human cancer cell lines (K562, A549, HCT116, PC3, U87-MG, MIA-Paca2, HEK293, NCI-N87, RT4, U2OS, A2780, MRC -5 and JIMT-T1) using a luminescence spectrophotometric method; hence, the oils showed significant cytotoxic activities. The antibacterial activities of the oils were assayed on five Gram-positive bacteria (Staphylococcus aureus, Enterococcus faecalis, Streptococcus agalactiae, Staphylococcus epidermidis and Corynebacterium sp.) and seven Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Shigella sonnei, Salmonella enterica sv. Typhimurium and Enterobacter cloacae) by agar disc diffusion methods. Both essential oils exhibited moderate activities against Streptococcus agalactiae, and thus each has an activity against Pseudomonas aeruginosa for EOOB and against Enterobacter cloacae for EOOA, while the others are negative. The findings of this work showed the promising anticancer effects of both oils against total cell lines with a potential for use as natural alternatives to synthetic drugs; however, there was only an antibacterial effect against Streptococcus agalactiae.