The antimicrobial activity of phenolic fraction of extra virgin olive oil was tested on the cell growth of Lactobacillus delbrueckii subsp. bulgaricus, Streptococcus thermophilus and Kluyveromyces marxianus and the mycelia growth of Penicillum frequentens in vitro. The phenolic extracts significantly inhibited the cell growth of S. thermophilus whereas the growth of Lb. delbrueckii subsp. bulgaricus was slightly affected in the presence of 100 mg L-1 phenolic extracts at pH 6.9. The phenolic extracts had a significant inhibitory activity against K. marxianus growth at pH 4 but not at pH 6.9 due to increased solubility of phenolics under acidic conditions (low pH). The growth of P. frequentens, expressed as mycelia wet weight was not greatly affected by the presence of phenolic extracts at varying concentrations at pH 4 and 6.9. It was concluded that the antimicrobial activity of phenolic extracts of extra virgin olive oil was dependent on concentration, pH, time and the type of micro-organism.