Roots and aerial parts of Cistaceae have been used since ancient times in the Mediterranean cultures for its medicinal properties. In this study, phenolic and tannin content of C. ladanifer and C. populifolius leaves aqueous extracts were determined and their antioxidant and antimicrobial activity were fully studied by several in vitro assays. Their major compounds were identified and quantitated by high-performance liquid chromatography with diode array detection coupled to electrospray ion-trap mass spectrometry. Cytotoxicity on a panel of human cancer cells was also determined. C. populifolius extract was stronger antioxidant than C. ladanifer extract in electron transfer reaction based assays but C. ladanifer extract was more effective to inhibit peroxyl radicals. The major compounds in both extracts were ellagitannins, especially punicalagins derivatives, showing C. populifolius a higher content. C. ladanifer showed noteworthy antibacterial activity against Staphylococcus aureus, whereas C. populifolius was effective against Escherichia coli, with MICs values of 154 and 123 microg/mL, respectively. Last, both extracts showed a notorious capacity to inhibit the proliferation of M220 pancreatic cancer cells and MCF7/HER2 and JIMT-1 breast cancer cells. The leaves of these plants suppose a source for water-soluble ellagitannins-enriched polyphenolic extracts with antioxidant and antimicrobial activities. Their cytotoxic activity against several cancer cells may deserve further attention.