The application of Low-Temperature Plasmas has shown great potential as an effective and alternative tool for microbial inactivation in recent years. Nevertheless, further investigations are required to fully understand the possible factors influencing these processes. The present study aims to investigate the effectiveness of square wave modulated Volume Dielectric Barrier Discharge on the direct inhibition of conidial germination in five different fungal species, various discharge conditions, and medium composition. The five different fungal species used were Botrytis cinerea, Monilinia fructicola, Aspergillus carbonarius, Fusarium graminearum, and Alternaria alternata. On water agar, the inhibition of Botrytis cinerea was influenced by the applied voltage, which mainly reflects the uniformity of the treatment. Under the selected voltage condition, the inhibition increased with treatment duration and decreased with fungal spore complexity. B. cinerea and M. fructicola, with unicellular conidia and low melanin content, showed similar behaviour and high sensitivity to the treatment. F. graminearum and A. alternata, both having multicellular conidia, were more resistant to the plasma treatment and showed different sensitivity likely due to different content in melanin. However, after 1 min of treatment, complete inhibition of conidial germination was achieved for all the tested species. Inhibition of A. carbonarius conidia on different agarized media containing dextrose or malt extract was influenced by the complexity and composition of the medium, being potato dextrose agar that more hindered the plasma efficacy. Each medium exhibited a different electrical response studied by Electrical Impedance Spectroscopy and morphology observed by Scanning Electron Microscopy images. These differences translated into a different response to the applied electrical field, influencing plasma generation and uniformity.