Abstract Background In our continuing search for biologically active natural enemies from North of Africa with special reference to Tunisian fungi, our teamwork screened fungi from different ecological habitats in Tunisia. Our previous study on the comparative effectiveness of filamentous fungi in the biocontrol of Meloidogyne javanica, a taxon (Lecanicillium) showed high potentiality against M. javanica. We undertook the present study to evaluate the ability and understand the mechanism of this fungal parasite as a biological control candidate against the root-knot nematode M. javanica. This study used in vitro bioassays with fungal filtrate cultures, scanning electron microscopy (SEM) observation, and isobaric tag for relative and absolute quantitation (iTRAQ) methodology to characterize the biological and molecular features of this fungus. Results The microscopic and SEM observation revealed that Lecanicillium sp. exhibited exceptional hyperparasitism against M. javanica eggs. The hyphae of this fungi penetrated the eggs, causing destructive damage to the outer eggshell. The exposure to five concentrations of Lecanicillium sp. filtrate cultures showed high inhibition of egg hatching, which increases depending on the exposure time; the best results are recorded at 50%, 75%, and 100% dilutions after seven days of exposure. The SEM observation of nematode-parasitized eggs and juveniles suggests that the production of lytic enzymes degrades the egg cuticle and fungal hyphae penetrate unhatched M.javanica juveniles. Forty-seven unique proteins were identified from the Lecanicillium sp. isolate. These proteins have signalling and stress response functions, bioenergy, metabolism, and protein synthesis and degradation. Conclusion Collectively, Lecanicillium sp. had ovicidal potentiality proved by SEM and proteomic analysis against root-knot nematode’ eggs. This study recommended applying this biological control candidate as a bio-agent on vegetable crops grown in situ.