Olive leaves represent a rich source of bioactive compounds, specially polyphenols that have the potential of application in the food and pharmaceutical industries. Industrial production of olive oil results in significant leaf residues, therefore there is a great interest to exploit the byproducts of production. Ultrasonic extraction is an effective and economically accessible method for extraction of biologically active compounds from plant material. Therefore, the aim of this study was to optimize the extraction of polyphenols from olive leaves by ultrasonic extraction and to assess antimicrobial activity and microbiological purity of extracts. Due to its high specificity, optimal extraction conditions were determined experimentally by examining different variables. Obtained values of amplitude (A=75%), mass (6 g/l) and time (t=3 min) were shown to be the most optimal. However, olive leaf extracts thus obtained did not show any antimicrobial activity. Modified sonication assisted extraction gave an olive leaf extract with higher concentrations of total polyphenols that showed antimicrobial activity with the bacteria Salmonella enterica subsp. enterica (DSM 14221), Escherichia coli (ATCC 25922) and Listeria innocua (ATCC 33090). Antimicrobial effect was obtained at concentrations 600 and 200 μg of total polyphenols, showing up to 37.0% efficiency of conventional antibiotics used in combating infections caused by Gram negative, and up to 32.6 % of the effectiveness of antibiotics to combat infections caused by Gram positive bacteria. Also, it was determined that aerobic mesophilic and sporogenic bacteria, bacteria from the Enterobacteriaceae family and fungi (mold and yeasts), form the natural microbiota of olive leaf. All extraction methods, sonication assisted, modified by ultrasound assisted and conventional extraction had bactericidal impact on the bacteria of the Enterobacteriaceae family and fungicidal impact on mold and yeast. On the other hand, neither conventional nor ultrasonic extractions affected the number of aerobic sporogenic bacteria.