Lettuce (Lactuca sativa L.) has been more and more present in the everyday nutrition in the last few years, with very characteristic seasonal consumption. This vegetable has a high prophylactic effect due to high level of biologically active matters in it. Its strong and visually attractive colours indicate the presence of phenol compounds and therefore the antimicrobial activity. Lettuce in this research has been grown in the winter in two types of controlled conditions: plastic and glass houses. The research material was the variety Neva belonging to olive-butter type of lettuce, originating from Institute for Vegetable Crops from Smederevska Palanka. At the horticultural maturity, lettuce samples have been tested for antibacterial activity. Extracts of lettuce leaves were prepared by cold maceration with 50% ethanol. In order to determinate the antimicrobial activity of the extract, clean cultures originating from The Institute of Virology, Vaccines and Sera Torlak: Staphylococcus aureus ATCC 25923, Klebsiella pneumoniae ATCC13883, Escherichia coli ATCC 25922, Proteus vulgaris ATCC13315, Proteus mirabilis ATCC14153, Bacillus subtilis ATCC6633, Candida albicans ATCC10231, Aspergillus niger ATCC16404 have been used. Antimicrobial activity was determined by using microdilution. Reference antibiotics used for bacterial strains have been Amracin and Nystatin for fungal strains. The research aimed to establish antimicrobial effect of lettuce extract originating glass house (S1) and plastic house (S2) in order to determinate the possibility of using these extracts in the pharmaceutical and food industry as a potential “natural preservative”. The results showed that the best antimicrobial activity had lettuce extract from S2 plastic house on strains Aspergillus niger and Bacillus subtilis in concentration 19,53 μg/ml while lettuce extract S1 from green house has been 78,125 μg/ml on all researched strains except on Proteus mirabilis and Staphylococcus aureus.