Germline TP53 pathogenic variants can lead to a cancer susceptibility syndrome known as Li–Fraumeni (LFS). Variants afecting its activity can drive tumorigenesis altering p53 pathways and their identifcation is crucial for assessing individual risk. This study explored the functional impact of TP53 missense variants on its transcription factor activity. We selected seven TP53 missense variants (c.129G>C, c.320A>G, c.417G>T, c.460G>A, c,522G>T, c.589G>A and c.997C>T) identifed in Brazilian families at-risk for LFS. Variants were created through site-directed mutagenesis and transfected into SK-OV-3 cells to assess their transcription activation capabilities. Variants K139N and V197M displayed signifcantly reduced transactivation activity in a TP53-dependent luciferase reporter assay. Additionally, K139N negatively impacted CDKN1A and MDM2 expression and had a limited efect on GADD45A and PMAIP1 upon irradiation-induced DNA damage. Variant V197M demonstrated functional impact in all target genes evaluated and loss of Ser15 phosphorylation. K139N and V197M variants presented a reduction of p21 levels after irradiation. Our data show that K139N and V197M negatively impact p53 functions, supporting their classifcation as pathogenic variants. This underscores the signifcance of conducting functional studies on germline TP53 missense variants classifed as variants of uncertain signifcance to ensure proper management of LFS-related cancer risks.