The aqueous extracts of Hibiscus calyphyllus (HcA), Hibiscus micranthus (HmA) and Hibiscus deflersii (HdA) growing in Saudi Arabia didn't receive enough phytochemical and biological studies. This inspired the authors to investigate the phytochemicals of these extracts for the first time using UPLC-ESI-MS/MS in negative and positive ionization modes. The analysis afforded the tentative identification of 103 compounds including phenolic compounds, flavonoids and anthocyanins. Moreover, in-vitro evaluations of their cytotoxic, antioxidant, antidiabetic and anti-obesity activities were examined. The results showed that aqueous extract of Hibiscus calyphyllus showed the highest activity as antioxidant agent (SC50 = 111 ± 1.5 µg/mL) compared with ascorbic acid (SC50 = 14.2 ± 0.5 µg/mL). MTT assay was used to evaluate cytotoxic activity compared to cisplatin. Hibiscus deflersii showed the most potent cytotoxic effect against A-549 (human lung carcinoma) with IC50 = 50 ± 5.1 μg/mL while Hibiscus micranthus showed a closed effect with IC50 = 60.4 ± 1.7 μg/mL. Hibiscus micranthus showed the most potent effect on HCT-116 (human colon carcinoma) with IC50 = 56 ± 1.9 μg/mL compared with cisplatin (IC50 = 7.53 ± 3.8 µg/mL). HcA and HdA extracts showed weak cytotoxic activity against A-549 and HCT-116 cell lines compared to the other extracts. Eventually, Hibiscus deflersii showed an astonishing antidiabetic (IC50 = 56 ± 1.9 µg/mL) and anti-obesity (IC50 = 95.45 ± 1.9 µg/mL) activities using in-vitro α-amylase inhibitory assay (compared with acarbose (IC50 = 34.71 ± 0.7 µg/mL)) and pancreatic lipase inhibitory assay (compared with orlistat (IC50 = 23.8 ± 0.7 µg/mL), respectively. In conclusion, these findings are regarded as the first vision of the phytochemical constituents and biological activities of different Hibiscus aqueous extracts. Hibiscus deflersii aqueous extract might be a hopeful origin of functional constituents with anticancer (on A-549 cell line), antidiabetic and anti-obesity activities. It might be a natural alternative remedy and nutritional policy for diabetes and obesity treatment without negative side effects. Isolation of the bioactive phytochemical from the aqueous extracts of aerial parts of Hibiscus calyphyllus, Hibiscus micranthus and Hibiscus deflersii and estimating their biological effects is recommended in further studies.