Protein aggregating is known as a leading pathogenic characteristic of a wide range of neurodegenerative diseases (NDs). Preventing amyloid-β (Aβ) aggregation and uncovering the associated mechanism through the application of small bioactive compounds can be considered as a useful strategy in hampering the onset of ND. In this study, we analyzed the inhibitory effects of cirsiliol, a trihydroxy-dimethoxyflavone, against human Αβ42 fibrillization. Also, we explored the probable neurotoxicity of Αβ42 oligomers grown with cirsiliol at different molar ratios on PC-12 cells after 24 h. The results showed that significant changes in ThT and ANS fluorescence intensities, Congo red absorbance, and ellipticity changes were modulated by co-incubation of cirsiliol with Αβ42, in a concentration-dependent manner. The spectroscopy outcomes were also supported by imaging analysis, where a few Αβ42 fibrillar conformations were detected with cirsiliol. In addition, cellular assays demonstrated that co-incubated Αβ42 samples with cirsiliol regulated the cell mortality, LDH release, and caspase-3 activation relative to the PC-12 exposed to Aβ42 oligomers alone. In conclusion, it can suggest that cirsiliol can be used as a potential candidate in the development of small molecules-based drugs for the advancement of therapeutic platforms against ND.