Honey has a history of medical use and is known as bio-alternative therapy. This research assessed the phytochemical and biological activity of the medical grade manuka honey (MH). Gas chromatography–mass spectrometry (GC–MS) was chosen to investigate bioactive compounds of the MH. The DPPH and ABTS free radical scavenging and beta-carotene antioxidant activities as well as the antibacterial and antibiofilm effects against S. aureus, B. subtilis, E. coli and P. aeruginosa were all determined. Furthermore, to gauge anticancer properties of MH, a MTT assay was opted towards three cell lines, including HCT-116 (colon), A549 (lung) and MCF-7 (breast) cancer cells. The GC–MS analysis of the tested MH revealed the identification of various chemical constituents belonging to the fatty acids, phenols, and esters. The MH was found to have higher reducing power activity (DPPH IC50: 7.36; ABTS IC50: 4.49 mg/mL) than the beta-carotene bleaching power (IC50: 37.51 mg/mL). Similarly, the MH was noted to be more active against the planktonic and biofilm of Gram-positive bacteria. Furthermore, a dose-dependent anticancer potential was observed, although a significant anticancer potential was pointed out against the MCF-7 and A549 cell conforming to the IC50 values of 9.05 and 9.37 mg/mL, respectively. This study’s results have highlighted the MH’s chemical composition with significant bioactivities.