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Stearic acid (SA) and oleic acid (OA) which are inherently existing fatty acids (FAs) in the body can alter cell membrane function and interact with each other. However, discrepancies arise as to whether these effects are beneficial or harmful on the body. To resolve this ambiguity, there is a dire need to study how FAs can affect the etiology of diseases and their treatment. In this study, we aimed to investigate long chain FAs aggregation behaviors and their effects on membrane integrity and cell viability. We determined the critical aggregation concentration (CAC) of SA and OA (1110 mu M and 300 mu M, respectively which were less amount than that used in nanocarriers). In TEM images, hexagonal overlapped or fused structures of SA were seen, whereas quite small spherical clusters of OA were obtained. Membrane integrity assessments demonstrated that SA and OA at their own CAC and below could crack the lipid junctions on membrane mimicking systems. Moreover, they completely disrupt the membrane integrity above the CAC at pH 7.2. Cell viabilities on various cell lines were assessed after exposed to SA or OA aggregates. SA was more aggressive than OA on cell death in all cell lines. The effect of SA on PC3 cell lines was in a concentration-dependent manner. The effect of SA above CAC boosted the inhibition of cell viability. Furthermore, OA showed a proliferation effect on PC3 cells. Consequently, the aggregation behavior of FAs should be considered as a noteworthy factor in physiological functions.