<p>This research focuses on the phytochemicals profiling and biological potential of selected olive cultivars from Punjab, Pakistan. Ultrasound-assisted aqueous ethanolic extraction was employed as a green route to extract diversified classes of bioactive components from five olive fruit cultivars: Ottobratica (V-1), Coratina (V-2), Picual (V-3), Koroneiki (V-4), and Arbequina (V-5). The extracts were purified via liquid-liquid partitioning and evaluated for antioxidant, antibacterial, hemolytic and anti-biofilm activities and further characterized for bioactives profiling using GC-MS. A total of 38 phytochemicals including 15 phenolics, 6 fatty acids, 2 esters, 7 heterocyclic, 2 alcohols, 4 aldo-keto, 1 oxysilane &amp; 1 hydrocarbon were identified. The V-3 olive cultivar exhibited the highest (<i>p</i> &lt; 0.05) contents of phenolics, flavonoids, alcoholic, and aldo-keto compounds, as well as strongest DPPH radical scavenging, and biofilm lysing activity as evidenced by percent component analysis and correlation studies. V-3 samples extract exhibited the least RBC percent hemolysis. The olive fruit crude extracts (OFCEs) and olive fruit phenolic enriched extracts (OFPEEs) from V-2 &amp; V-3 exhibited superior anti-bacterial activity against Gram-negative <i>Escherichia coli</i> (<i>E. coli</i>) and Gram-positive <i>Staphylococcus aureus</i> (<i>S. aureus</i>). Molecular docking data showed that <i>β</i>-sitosterol, quercetin, squalene, and 1’-hydroxy-4,3’-dimethyl-bicyclohexyl-3,3’-dien-2-one, although present in small amount, are the best ligands with potent binding affinities, supporting the antibacterial potential of the tested olive fruits extracts against <i>S. aureus</i> and <i>E. coli</i>. The phytochemicals profiling and biological activities data of the present study can be valuable towards exploring the functional food and nutra-pharmaceutical applications of these newly developed Pakistani olive cultivars.</p>