Dietary fatty acids function in many processes that impact cardiovascular health. Specifically, polyunsaturated fatty acids (PUFAs), are expected to improve cardiovascular function through the alteration of membrane properties of diverse cell types. Global membrane properties have increasingly been shown to impact the activity of distinct mechanosensitive membrane proteins. However, the mechanism by which these properties affect mechanosensitive channel activity remains unclear. PUFAs are hypothesized to alter bilayer mechanical properties but monoacyl fatty acids have been studied sparingly in phospholipid bilayers. Moreover, the effect of monoacyl fatty acid chain length, unsaturation, and location of unsaturation on membrane mechanical properties have not yet been determined. With these questions in mind, we incorporated monoacyl PUFAs of different chain lengths and degrees of unsaturation into phospholipid bilayers. Using micropipette aspiration and fluorescence anisotropy, we measured the effect of various PUFAs on the membrane area expansion modulus and fluidity, respectively, of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) vesicle membranes. By systematically altering the identity of fatty acids, we observed that long-chain omega-3 fatty acids—but not monounsaturated fatty acids—reduce the membrane area expansion modulus and increase the fluidity of DOPC vesicle membranes. In addition, we observe that two major omega-3 fatty acids, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), impact the area expansion modulus and fluidity of DOPC membranes containing cholesterol domains in different ways. These results provide insight into how EPA and DHA may have distinct effects on the bilayer properties of mammalian cells, despite being chemically similar. Our results demonstrate several ways in which dietary monoacyl fatty acids impact the mechanical properties of phospholipid bilayers and present a potential route through which fatty acid uptake into cell membranes may influence the activity of membrane proteins.