Since the dawn of civilization, people have turned to plants as a safe and efficient form of treatment for a variety of diseases. It has long been known that Calotropis procera has the potential to treat a number of diseases. In this study, the C. procera leaf aqueous extract was obtained using the maceration method, and p-coumaric was found to be the main compound. The extract was rich in phenols (174.82 mg gallic acid equivalent/g) and flavonoids (1781.7 µg quercetin equivalent/g). The extract had high antioxidant properties, as indicated by the IC50 values obtained for 2,2-diphenyl-1-picrylhydrazyl (DPPH) (366.33 μg/mL) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS) (169.04 μg/mL), as well as the ferric ions reducing antioxidant power (FRAP) (1.67 μg ascorbic acid equivalent/g of the extract). The cytotoxicity of the extract was evaluated against the survival of HT 29 cells, and the IC50 was found to be 236.87 μg/mL. The most resistant and sensitive strains to the extract were Escherichia coli and Staphylococcus aureus, respectively. The morphological changes of these strains were demonstrated through scanning electron microscopy and confocal laser scanning microscopy. The C. procera extract could be therefore used as an antioxidant, antimicrobial, and anticancer agent.