Kalanchoespecies are succulents occurring in tropical regions. They have many biological and pharmacological properties. In this study, the cytotoxic and antimicrobial activities of water and dichloromethaneKalanchoefractions obtained from ethanol extracts of threeKalanchoespecies—K. daigremontiana,K. pinnata, andK. blossfeldianawere estimated. The cytotoxic effect was assessed on human cancer cell lines—ovarian SKOV-3, cervical HeLa, breast MCF-7, and melanoma A375—by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The antimicrobial activity was estimated on selected Gram-positive and Gram-negative bacteria strains and onCandida albicans. The phytochemical analysis of selectedKalanchoeextracts was conducted by LC-QTOF-MS. The obtained results showed that the water fraction ofK. blossfeldianawas active both on the tested cancer cells (IC50values were 28.28 ± 2.76 and 32.51 ± 0.69 µg/mL on HeLa and SKOV-3, respectively) and bacteria strains (MIC values were 16 and 32 µg/mL onS. epidermidisandS. aureus, respectively). The water fraction ofK. pinnataalso had a significant effect onS. epidermidisandS. aureus, with MIC values of 32 and 64 µg/mL, respectively. The water fraction ofK. blossfeldianatriggered a decrease in mitochondrial membrane potential (MMP) and induced cell cycle arrest in the G2/M phase in the SKOV-3 and HeLa cells. This fraction did not significantly increase cellular oxidative stress level. The DPPH and ABTS assays revealed that the water fraction ofK. blossfeldianahad a strong antioxidant effect (IC50was 9.44 ± 0.06 and 3.17 ± 0.1 µg/mL, respectively). The phytochemical analysis of the extracts ofK. blossfeldianaandK. pinnatarevealed the presence of at least 218 main components. The most frequently occurring were flavonol glycosides (31 metabolites), phenylpropanoids (13 metabolites), gallic acid derivatives (13 compounds), benzoic acid derived compounds (14 metabolites), and acyclic alcohol glycosides (16 compounds). In addition, proanthocyanidins were detected mainly inK. blossfeldiana.The study indicates that the water fraction ofK. blossfeldianahas significant biological potential and can be further investigated towards anticancer and antimicrobial application.