Biofuels are a promising alternative to substitute petroleum-based transportation fuels and could contribute to sustainable development. This work studies the composition and properties of the fatty acid methyl esters (FAMEs) obtained from Citrullus colocynthis, a plant that grows in arid and dry conditions. Samples from seven locations in Tunisia were collected to study the possible variability in their composition and properties to assess the performance in a combustion engine. Results show suitable properties and future studies are encouraged to assess the potential productivity at a large scale, regarding that domestication of the plant could contribute to improving its quality and productivity.