The LC-MS analysis of the MeOH extract of the aerial parts of Pergularia tomentosa led to the isolation of 23 compounds, of which the structures were elucidated unambiguously by NMR spectroscopic data analysis. Three new doubly linked cardenolides (4, 13, 14) along with several known cardenolides (1-3, 5, 7, 8, 15-23) and flavonol glycosides (6, 9-12) were identified. LC-HRESIMS analysis, in the negative-ionization mode, showed the absence of flavonoids in a methanol extract of the roots of P. tomentosa. On the basis of the antiproliferative activity reported for cardenolides, the isolated compounds were tested for their ability to decrease the cell viability of five different human cancer cell lines, PC3, HeLa, Calu-1, MCF-7, and U251MG, exhibiting IC50 values ranging from 0.2 to 8.0 μM. Moreover, an S-phase entry assay was performed to investigate if the compounds could affect the cell cycle progression of PC3 prostate carcinoma cells. The results obtained demonstrated that the compounds 4, 7, and 14 at 1 μM considerably reduced the number of cells in the S-phase.