Unsaturated fatty acids and esters can be oxidized in situ during ionization using a low temperature plasma (LTP) probe. The discharge generates ozone from air that reacts with and cleaves olefins. The molecular ions of the resulting acid/ester oxidation products are present in the full scan mass spectra and are confirmed by exact mass measurements. The fragmentation information can be used to assign double bond positions. We have successfully applied this strategy to a range of mono-/polyunsaturated fatty acids and fatty acid methyl/ethyl esters to assign their double bond locations. The procedure allows rapid and direct identification of double bond positions in situ at atmospheric pressure without sample preparation prior to mass spectrometric analysis. Microbial fatty acid ethyl ester (FAEE) mixtures from complex bacterial samples were directly analyzed by this method. Structural confirmation of their diagnostic ions by using exact mass measurements and tandem mass spectrometry confirms double bond positions in unsaturated bacterial FAEEs.