Grapevine Fanleaf Virus (GFLV) is one of the diseases that causes major losses in grape cultivars in Chile. The main vector described for this virus is the nematode Xiphinema index. Pseudomonas strains have been already used as bio-control agents for fungi, bacteria and nematodes and the activity reported to be mediated by the production of antibiotics such as 2,4-diacetylphloroglucinol (Phl). In the present work, new local P. fluorescens isolates were obtained, characterized and assessed for their antagonistic activity against X. index. These new isolates were compared to the highly active Pseudomonas strain CHA0 by in vitro challenges in which whole bacterial extracts were produced by culturing in King B-agar dishes. Results showed that 3 h after interaction between extracts and X. index, the isolates RE4 and CHA0 produced 100% of dead nematodes. Application of bacterial supernatants reported the same efficacy in both CHA0 and RE4, leading to 100% of dead X. index individuals after three hour of treatment. Conversely to CHA0, no Phl antibiotic was detected in RE4 cultures or supernatants, suggesting a different mechanism for this antagonism. RE4 is proposed as a new Pseudomonas fluorescens isolate with nematicide activity which is not mediated by Phl production and that is able to control X. index under in vitro assays.