The bioactive Hemp Seed Oil (HSO) is becoming very popular in the medical and research fields due to its antimicrobial properties against several diseases caused by bacteria and fungi. However, the effect of hemp-growing factors and extraction methods on the bioactivity of HSO does not receive adequate research attention. Therefore, this review aims to investigate the effect of growth factors and extraction methods on the antimicrobial activity of HSO. Articles were retrieved from Google Scholar and the Scopus database and screened against inclusion and exclusion criteria. The study revealed that HSO prefers warm climates and favorable humidity ranging from 20 to 39 °C and 79–100% per year, respectively, and rainfall of 324 mm daily. The multivariate linear regression shown excellent prediction (R2 = 0.94) with climates upon Zone of Growth Inhibition (ZGI) of Gram-positive bacteria. Temperature is the strongest predictor (p < 0.01) followed by humidity and rainfall (p < 0.05). Furthermore, well-drained loam soil rich in organic matter seems to stimulate the antimicrobial activity of HSO. The major constituents that influence HSO’s antimicrobial ability to Staphylococcus aureus were cannabidiol (CBD), β-caryophyllene, and limonene. The extraction methods showed less influence on the HSO bioactivity. HSO did not show significant antioxidant activity, but Hemp Seed Hull (HSH), Hemp Seed Flour (HSF), and Hydrolyzed Hemp Seed Protein (HPH), expressed promising DPPH scavenging ability.