Cancer research has made significant progress in recent years, particularly with the application of new omics technologies. By addressing proteomics, it is possible to gather vast amounts of data that offer a comprehensive view of the molecular mechanisms involved in potential treatments. This research aims to use label-free quantitative proteomics coupled with pathways and networks-based bioinformatics analysis, to uncover the molecular mechanisms of the natural alkylphenol Gibbilimbol B isolated from P. eriopodon. Dynamic proteomic profiling of breast tumor cell lines treated with Gibbilimbol B showed the impact of several molecular pathways in a time and cell-dependent manner. The activation of the apoptotic pathway was confirmed for both MCF-7 and MDA-MB-231 cells, nevertheless, the proteomic profile provided new insights into molecular mechanisms triggered by ubiquitin-dependent protein catabolic process and the identification of P53-Independent DNA damage checkpoint specific for MCF-7 cells among others. On the contrary, in MDA-MB-231, the pattern of the protein profile was correlated with a possible response to endoplasmic reticulum stress, the activation of nuclear receptors in response to pro-inflammatory processes, and an immune response related to antigen processing. Overall, these advances provide detailed insights at the cellular level for understanding potential treatment strategies.