The soybean, Glycine max, is a major agricultural commodity in Brazil, significantly affected by a wide range of plant- parasitic nematodes, notably Aphelenchoides besseyi. With no resistant cultivars available or registered nematicides, management options for this nematode are limited. This study investigated the effects of cell-free culture filtrates from three Purpureocillium lilacinum isolates (CCT 7766, UEL PAE 10 and PLI) on A. besseyi inactivity, mortality and reproduction in vitro compared to a water control. Nematodes were treated with P. lilacinum culture filtrates, and their activity was quantified after 24, 48 and 72 h. Mortality was assessed by counting inactive individuals after the addition of a Na2CO3 solution. Reproduction effects were tested by treating nematodes with filtrates and culturing them on Fusarium sp. plates for 21 days. After this period, nematodes that migrated to the lids of the plates were counted under a microscope (100x magnification) to determine the final nematode population. Results indicated 100% reduction in active nematodes across all isolates after 24 h. Culture filtrates exhibited low nematicidal effects, with nematode motility resuming after Na2CO3 exposure. Interestingly, individuals exposed to the CCT 7766 culture filtrate displayed a distinctive phenotype, characterised by impaired motility and partial body paralysis, even after the recovery period. Regarding reproduction, PLI strain significantly reduced the final nematode population - and consequently the reproduction factor - when compared to the water control treatment. This study validates a suitable methodology for effectively assessing the mortality of A. besseyi in vitro, and suggests P. lilacinum as a promising biocontrol agent, although further research is needed to explore its efficacy in agricultural settings.