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Olive oil isolated from the fruits of the Olea europaea L. is an important part of Mediterranean diet. It is known for its diverse biological actions. Furthermore, a little amount of fixed oil and other bioactive components can also be extracted from the olive seeds which are considered as byproduct of olive oil extraction. Therefore, this study was designed to analyze the fatty acid composition and to perform in vitro biological studies on fixed oil isolated from olive seeds. The fixed oil was isolated from the olive seeds of Syrian and Greek black olive fruits by using Soxhlet apparatus. The purity was checked by measuring its refractive index. Composition of two isolated oils and a commercially available virgin olive oil was determined by preparing their Methyl esters (FAME) followed by GC-MS analysis. Various in vitro assay methods were used to investigate activities such as antioxidant activity by 1,1-diphenyl-2-picrylhydrazyl (DPPH), cytotoxic activity by using Brine shrimps lethality bioassay and antimicrobial activity against two bacterial strains; Staphylococcus aureus and Escherichia coli by agar well diffusion method. GC-MS analysis revealed that the two isolated oils differ quantitatively in chemical composition with oleic acid identified as the major chemical constituent (62.6% and 73.56%). Both the fixed seed oils showed a concentration dependent DPPH radical scavenging activity ranging from 8 to 76% inhibition. The oils also exhibited excellent cytotoxic activity but no antimicrobial activity was observed. The chemical composition of the isolated fixed olive seed oil is found to be almost similar to the commercially available fruit olive oil. The fixed oil from the seeds of olive fruits possesses useful biological actions. Further studies are needed to isolate and quantify their bioactive constituents.