Fusarium oxysporum is a widely distributed phytopathogen that affects agricultural crops and has demonstrated relevance in human and veterinary medicine. The virulence of this fungus involves factors such as mycotoxins, immunomodulatory proteins, and the ability to form biofilm. We assessed the pathogenicity of F. oxysporum in its planktonic form, as well as the influence of the extracellular matrix (ECM) from its biofilm on T. molitor larvae and cell culture. F. oxysporum inoculum was injected into larvae at different concentrations, and the survival curve was observed over 10 days. To evaluate the effects of ECM components, biofilms of 24, 72, 96, and 168 h of maturation were used. After extracting the ECM from these biofilms, it was injected into the larvae to assess the hosts response. For the cytotoxicity test of the ECM, were used on Vero cells. The increase in fungal inoculum concentrations was directly proportional to the larval mortality rate. When larvae were infected with the ECM, there was a 40% mortality rate and signs of weakness in the surviving larvae. Furthermore, biofilm metabolites showed cytotoxic effects, with reductions in cellular activity ranging from 20 to 49%. This alternative host model proved effective in investigating the fungal pathogenicity. Additionally, ECM components negatively affected cell viability, suggesting their importance in the damage caused by the fungus in host tissues. This study provides insights for the first time into F. oxysporum virulence and highlights the importance of considering the biofilm ECM in the context of fungal infections.