Several nonpathogenic strains of Fusarium oxysporum have been selected as potential biological control agents. Culture filtrate of F. oxysporum f. sp. ciceris was extracted sequentially with ethyl acetate (EtOAc) and n-butanol (n-BuOH). Chromatographic separation of EtOAc concentrate has afforded five compounds, bikaverin (1), 3-O-methyl-8-O-methyl fusarubin (2), 8-O-methyl fusarubin (3), anhydrofusarubin (4) and fusarubin (5). Structures of these compounds were elucidated by detailed NMR and LC/ESI-MS spectra. Anti-nemic activity of the extracts and secondary metabolites were evaluated against root-knot nematode, Meloidogyne incognita and reniform nematode, Rotylenchulus reniformis. EtOAc concentrate was found to be strongly active against M. incognita (LC50 56.2μgmL−1) followed by n-BuOH concentrate (LC50 97.4μgmL−1). However, these concentrates were moderately effective on R. reniformis (LC50 134.5–189.2μgmL−1). Fusarubin (LC50 248.9μgmL−1) was found to be highly active followed by anhydrofusarubin (LC50 257.6μgmL−1) against M. incognita. Structure anti-nemic activity relationship indicated comparatively more polar molecule with naphthoquinone nucleus as most active. Fusarubin analogues could be exploited further to develop standard nematicidal liquid formulation.