Abstract BACKGROUND Symbiotic bacteria from the genera Xenorhabdus and Photorhabdus associated with Steinernema and Heterorhabditis entomopathogenic nematodes (EPNs), respectively, show nematicidal properties against plant‐parasitic nematodes. This study investigated the diversity of EPNs in Floridian turfgrass systems and the effect of secondary metabolites of their symbiotic bacteria against grass root‐knot ( Meloidogyne graminis ) and sting ( Belonolaimus longicaudatus ) nematodes, major pests in turfgrass systems. RESULTS Six isolates of H. indica , four of S. glaseri , and two of S. diaprepesi were isolated and identified molecularly from three turfgrass locations. Additionally, their symbiotic bacteria, X. poinarii , X. griffiniae , X. doucetiae , X. indica, P. akhurstii and P. luminescens were isolated and molecularly identified. Nematicidal efficacy of bacterial secondary metabolites (crude extracts) was tested against M. graminis and B. longicaudatus nematodes in vitro . Bioassays demonstrated a concentration‐dependent increase in nematode mortality. The isolates X. poinarii 733 and X. griffiniae 1050 exhibited high mortality against M. graminis after 24, 48 and 72 h of exposure at 25% concentration (>87%) ( P < 0.05). In addition to these isolates, two others, P. akhurstii 846 and P. luminescens 1189, exhibited high mortality (44–100%) against B. longicaudatus after 72 h at 25%, 50% and 100% concentrations ( P < 0.05). CONCLUSION These findings indicate that Florida turfgrass soils harbor a high diversity of EPNs and associated bacteria. Furthermore, our results suggest for the first time that Xenorhabdus and Photorhabdus ‐derived secondary metabolites offer a promising biocontrol approach for managing M. graminis and B. longicaudatus . © 2025 Society of Chemical Industry.