Phoenix dactylifera, commonly called date palm, has great importance as a fruiting plant. The hot methanolic extract of date seeds (HMEDSE), was further fractionated into three fractions (F1, F2, and F3) through column chromatography. The three fractions were composed of various bioactive constituents which was analysed through GC-MS and FT-IR analysis. The results revealed remarkable antibacterial properties of crude HMEDSE against various pathogenic microorganisms affecting humans. The spectrum of activity of HMEDSE against various human pathogenic bacteria showed the following sequence based on its efficacy, Escherichia coli (17.6 ± 2.5 mm), Klebsiella pneumoniae (16.3 ± 2.5 mm), Staphylococcus aureus (16.3 ± 1.5 mm), Streptococcus pyogenes (15 ± 2.6 mm), Pseudomonas aeruginosa (15 ± 2 mm), and lastly, Bacillus subtilis (14.3 ± 2 mm). Furthermore, HMEDSE exhibited cytotoxicity, with an IC50 of 73.5 ± 0.5 µg/mL against MCF-7 ATCC breast cancer cells, leading to gradual apoptosis.