After decades of utilization of fossil-based and environmentally hazardous compounds for wood preservation against fungal attack, there is a strong need to substitute those compounds with bio-based bioactive solutions, such as essential oils. In this work, lignin nanoparticles containing four essential oils from thyme species (Thymus capitatus,Coridothymus capitatus,T. vulgaris, andT. vulgarisDemeter) were applied as biocides in in vitro experiments to test their anti-fungal effect against two white-rot fungi (Trametes versicolorandPleurotus ostreatus) and two brown-rot fungi (Poria monticolaandGloeophyllum trabeum). Entrapment of essential oils provided a delayed release over a time frame of 7 days from the lignin carrier matrix and resulted in lower minimum inhibitory concentrations of the essential oils against the brown-rot fungi (0.30–0.60 mg/mL), while for the white-rot fungi, identical concentrations were determined compared with free essential oils (0.05–0.30 mg/mL). Fourier Transform infrared (FTIR) spectroscopy was used to assess the fungal cell wall changes in the presence of essential oils in the growth medium. The results regarding brown-rot fungi present a promising approach for a more effective and sustainable utilization of essential oils against this class of wood-rot fungi. In the case of white-rot fungi, lignin nanoparticles, as essential oils delivery vehicles, still need optimization in their efficacy.