Owing to the key role of trehalose in pathogenic organisms, there has recently been growing interest in trehalose metabolism for therapeutic purposes. Trehalose-6-phosphate phosphatase (TPP) is a pivotal enzyme in the most prominent biosynthesis pathway (OtsAB). Here, we compare the enzyme characteristics of recombinant TPPs from five important nematode and bacterial pathogens, including three novel members of this protein family. Analysis of the kinetics of trehalose-6-phosphate hydrolysis reveals that all five enzymes display a burst-like kinetic behaviour which is characterised by a decrease of the enzymatic rate after the pre-steady state. The observed super-stoichiometric burst amplitudes can be explained by multiple global conformational changes in members of this enzyme family during substrate processing. In the search for specific TPP inhibitors, the trapping of the complex conformational transitions in TPPs during the catalytic cycle may present a worthwhile strategy to explore.