In the present study, the antimicrobial and cytotoxic activities, as well as the fatty acids composition in vegetable seed oils from linseed, purslane, luffa, and pumpkin were evaluated. For this purpose, two linseed oils and one luffa oil were commercially obtained, while purslane and pumpkin oils were obtained from own cultivated seeds. The results showed a variable fatty acids composition among the tested oils, with α-linolenic, linoleic, oleic, palmitic, and stearic acid being the most abundant compounds. In regards to particular oils, linseed oils were a rich source of α-linolenic acid, luffa and pumpkin oil were abundant in linoleic acid, while purslane oil presented a balanced composition with an almost similar amount of both fatty acids. Luffa oil was the most effective against two of the tested cancer cell lines, namely HeLa (cervical carcinoma) and NCI-H460 (non-small cell lung cancer), while it also showed moderate toxicity against non-tumor cells (PLP2 cell line). Regarding the antibacterial activity, linseed oil 3 and pumpkin oil showed the highest activity against most of the tested bacteria (especially against Enterobacter cloacae and Escherichia coli) with MIC and MBC values similar to the used positive controls (E211 and E224). All the tested oils showed significant antifungal activities, especially luffa and pumpkin oil, and for most of the tested fungi they were more effective than the positive controls, as for example in the case of Aspergillus versicolor, A. niger, and Penicillium verrucosum var. cyclopium. In conclusion, the results of our study showed promising antimicrobial and cytotoxic properties for the studied seed oils which could be partly attributed to their fatty acids composition, especially the long-chain ones with 12–18 carbons.