Cultivation of seedlings in trays requires the use of specifically developed substrates. This study presents the results of the analyses of selected physical and mechanical parameters of a peat–perlite substrate, in which seedlings of the Scots pine (Pinus sylvestris L.), Norway spruce (Picea abies (L.) H. Karst), European beech (Fagus sylvatica L.), and pedunculate oak (Quercus robur L.) were grown during a production season. For each species, the substrate parameters changed throughout the production season, substrate dry weight decreased, whereas its compactness increased with time. Independent of the species, the bulk density and total porosity of the substrate changed or deviated from the optimum range, although the values of porosity were close to maximum and those of density were close to the minimum. In certain periods in the V265 trays with beech and oak seedlings, the substrate was characterized by very low water capacity and excessive air capacity. Compactness measured with a cone penetrometer showed, that this parameter might be used for monitoring the substrate properties.