Propolis is mainly composed of plant resins, and its type is named according to the primary plant origin in its composition. Identification of propolis botanical origin is essential for predicting and repeating its pharmacological activity because of the variations in chemical composition. This study aimed to compare chemical composition of black poplar (Populus nigra L.) type-propolis (PR1 and PR2) and Eurasian aspen (P. tremula L.)-type propolis (PR3) by liquid chromatography-tandem mass spectrometry (LC-MS/MS) technique and to evaluate their biological activity profiles. According to LC-MS/MS results, in addition to marked caffeic acid phenethyl ester content in PR1 and PR2, flavonoid aglycones such as pinocembrin, chrysin, pinobanksin, and galangin were found to be dominant in these samples. On the other hand, PR3 contained relatively high concentrations of phenolic acids such as ferulic acid, p-coumaric acid, and trans-cinnamic acid. The anti-estrogenic activity test showed that PR2 exerted the highest anti-estrogenic activity by inhibiting cell proliferation by 44.6%. All propolis extracts showed anticancer activity, which was justified by decreasing activity on the 3D spheroid size in a concentration-dependent manner. Besides, all extracts showed moderate or potent antimutagenic activity in Salmonella typhimurium TA98 and TA100 strains with and without metabolic activation, respectively. In addition, the Comet assay results revealed that propolis extracts have a geno-protective effect against HO-induced DNA damage in CHO-K1 cells at 0.625 and 1.25 μg/mL concentrations. Overall, the result of this study may help in preparing standardized propolis extracts and developing products with defined pharmacological benefits in the food supplements industry.