Combating soil pathogens that disable plant emergence is among the most difficult challenges of global agriculture. Legumes, preferred in sustainable cultivation systems, are particularly sensitive to pre-emergence damping-off of seedlings. Seed dressing is therefore a very important element in the cultivation technology. The aim of this study was to compare the impact of biological (Pythium oligandrum) and chemical (carboxin + thiuram) seed dressing on the quantitative and qualitative composition of microorganisms participating in the epidemiology of this disease, under specific hydrothermal conditions and chemical properties of the soil (pH, humus, macro-, and micronutrient). Microorganism identification was done using the MALDI-TOF MS (Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry) technique. Species were assigned to frequency groups, and populations of pathogens, saprophytes, and antagonists were identified. The biodiversity of these communities was expressed with Simpson’s Reciprocal, Shannon–Wiener, and Evenness (Shannon) indices. In individual variants of seed pre-treatment, the correlations between individual edaphic factors and the suppression of pre-emergence damping-off, the number of isolates obtained from infected seedlings, and the share of individual trophic groups of fungi were assessed. The main causes of pre-emergence damping-off of broad bean seedlings are Ilyonectria destructans, Globisporangium irregulare, Fusarium equiseti, Rhizoctonia solani, and Fusarium solani. Eliminating seed treatment results in a seedling mortality rate of 33.5–42.5%. The effectiveness of the chemical protection product is 44.2% and 25.9%. Carboxin and thiuram reduce the diversity of microorganisms involved in the pathogenesis of pre-emergence damping-off and limit the presence of antagonistic fungi. Under the influence of P. oligandrum, there was a five-fold increase in the population of antagonists. An increase in humus in the soil reduces the percentage of diseased broad bean seedlings.