Measuring the efficacy of cultures applied at contaminanted sites for enhanced biological cleanup (bioremediation) is an important requirement for demonstrating the utility of bioaugmentation strategies. Current methods measure either the presence/absence of the applied bacteria or the removal of the chemical of interest. Peptide mass fingerprinting (PMF) is a mass spectrometry technique whereby proteins are cleaved at specific, predictable sites by proteases, and the resultant short amino acid sequence fragments are analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The technique is promising for microbial identification and phenotypic characterization of degadation activity. To assess the broader applicability of this unproven tool, commonly employed bioremediation agents (including Pseudomonas putida F1, P. sp. D5, Burkholderia sp. JS150, B. sp. DNT, Comamonas sp. JS765, Methylobium petroleophilum PM1, and Sphingomonas wittichii sp. RW1) were grown in pure culture on non-selective and selective substrates and analyzed by PMF. Sonicated biomass was processed by centrifugation, and the soluble cell lysate fraction was analyzed using MALDI-TOF MS following overnight digestion with trypsin. Spectra were acquired and searched against the NCBInr database or subsets thereof using the Mascot search tool (http://www.matrixscience.com). Analysis by PMF resulted in the identification of both targets, i.e., microorganisms and their key catabolic biomarkers, the latter including species-specific oxygenases responsible for the initial oxygenolytic activation of toluene, 3-phenoxybenzoic acid and dibenzofuran. Results suggest a broad applicability of PMF using MALDI-TOF MS for identifying bioremediation agents and assessing their degradation potential for pollutants of interest.