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Hartmannella vermiformis and Acanthamoeba polyphaga are frequently isolated from drinking water and permissive to Legionella pneumophila parasitization. In this study, extracellular factor(s) produced by H. vermiformis and A. polyphaga were assessed for their effects on cultivability of L. pneumophila. Page's amoeba saline (PAS) was used as an encystment medium for H. vermiformis and A. polyphaga monolayers, and the culture supernatants (HvS and ApS, respectively) were assessed against L. pneumophila growth. Compared to PAS and ApS, HvS significantly inhibited L. pneumophila strain Philadelphia-1 (Ph-1) cultivability by 3 log(10) colony forming unit (CFU) mL(-1) after 3 days of exposure compared to < 0.5 log(10) CFU mL(-1) reduction of strain Lp02 (P < 0.001). Flow cytometric analysis revealed changes in the percentage and cultivability of three bacterial subpopulations: intact/slightly damaged membrane (ISM), undefined membrane status (UD), and mixed type (MT). After 3 days of HvS exposure, the MT subpopulation decreased significantly (31.6 vs 67.2 %, respectively, P < 0.001), while the ISM and UD subpopulations increased (+26.7 and +6.9 %, respectively) with the ISM subpopulation appearing as viable but nonculturable (VBNC) cells. HvS was separated into two fractions based on molecular weight, with more than 99 % of the L. pneumophila inhibition arising from the < 5 kDa fraction (P < 0.001). Liquid chromatography indicated the inhibitory molecule(s) are likely polar and elute from a Novapak C18 column between 6 and 15 min. These results demonstrate that H. vermiformis is capable of extracellular modulation of L. pneumophila cultivability and probably promote the VBNC state for this bacterium.