This study aims to explore the synergistic effects of co-inoculation with Fusarium solani (F.s), Fusarium oxysporum (F.o), and Penicillium chrysogenum (P.ch) to enhance the digestibility and quality of lignocellulosic biomass for ruminant feeding. Wheat straw (WS), olive pomace (OP), and cedar wood (CW) were assessed as substrates. Results indicated varying impacts on lignin loss (L_loss), cellulose improvement (C_imp), and In Vitro True Digestibility improvement (IVTD_imp). F.o and P.ch co-inoculation exhibited the highest mean L_loss (53.74%), surpassing F.s and P.ch co-inoculation (18.23%) and F.s and F.o co-inoculation (19.23%). F.o_P.ch co-inoculation notably increased cellulose content (C_imp = 29.86 ± 18.19%) and IVTD_imp (40.74% ± 20.51%), while F.o_F.s showed minimal IVTD_imp (0.14 ± 11.42%). Substrates differed in fiber change and dry matter loss, with OP having the highest C_imp (25.6 ± 20.7%). Treatment duration influenced L_loss and IVTD_imp, increasing from 4 to 12 weeks. Co-inoculating F.o and P.ch enhances lignin degradation and biomass digestibility, improving their suitability for ruminant feed. Thoughtful selection of fungal combinations is crucial for optimizing co-inoculation. These findings support the utilisation of lignocellulosic biomass in ruminant feed.