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The aim of this study was to determine the antimicrobial activity of essential oils obtained from sweet basil (<i>Ocimum basilicum</i> L. cv. ‘Genovese’) cultivated in the open field under different shading conditions (red, blue, and pearl nets with a shade index of 50% and full sunlight exposure (control plants)), harvested at different times. The antimicrobial activity of basil essential oils (BEOs) obtained from all samples was determined for four microorganisms, while determinations for an additional five microorganisms included samples from non-shaded plants, plants grown under red and pearl nets, and second harvest of plants grown under blue net. Basil essential oil exhibited antimicrobial activity surpassing the activity of relevant commercial antibiotics regardless of growing conditions in the case of <i>B. cereus</i>, <i>K. pneumoniae</i> and <i>C. albicans</i>, while superior antimicrobial activity was exhibited in the case of essential oils from plants grown under blue nets in the case of <i>S. aureus</i>, <i>E. coli</i> and <i>P. vulgaris</i>. The influence of the application of colored shading nets was highly significant (<i>p</i> < 0.01) in the cases of all analyzed microorganisms except <i>C. albicans</i> and <i>P. aeruginosa</i>, while the influence of harvest time was proven in the cases of all microorganisms except <i>K. pneumoniae</i>. ANOVA proved that antimicrobial activities are highly dependent on the methods of plant production, shading treatment, and harvest time. Obtained results are discussed in relation to previously determined composition and yield of essential oils from basil grown under shade nets and harvested in different periods.