Currently, there is an increasing emphasis on the use of renewable sources. The Czech Republic is no exception. There has been an increase in the number of biogas plants and composting plants. The main purpose of these facilities is to process organic wastes of various origins in order to utilize the waste, both for energy production and as soil fertility enhancers. Last but not least, the biogas station produces digestate, which could be further utilized. The present work deals with the assessment of the effect of digestate (doses of 220; 150 and 80 kg N ha−1), mineral fertilizer (150 kg N ha−1), and compost application (150 kg N ha−1) on selected indicators of soil environment: (a) soil microbial activity (basal and substrate-induced respiration); (b) plant nutrient content (P, K, Ca, Mg); (c) leaching mineral N from the soil; and (d) production of plant biomass of model plant Lactuca sativa L. Furthermore, the work aims to assess the individual doses of digestate and to determine the optimal one. The above effects were studied within a pot experiment for 63 days in controlled conditions. There were significant differences in Nmin leaching, which was significantly the highest in the variant with the application of a mineral fertilizer and lowest in variants with the application of digestate (variant 150 and 80 kg N ha−1) and compost (150 kg N ha−1). In terms of soil microbial activity, a significant effect of digestate application (150 kg N ha−1) on basal respiration versus the mineral fertilizer application was found (difference > 20%). This dose of digestate also had the highest positive effect on substrate-induced respiration, proven against all other variants, except for the digestate application at 220 kg N ha−1. Particularly, as compared with the mere application of mineral fertilizer, the application of digestate dose at 150 kg N ha−1 had the best positive effect on plant biomass production (+20%). On the other hand, compost application most positively affected the increase in available contents of nutrients and thus long-term soil fertility. Therefore, the dose of 150 kg N ha−1 digestate may represent an interesting alternative to increase actual soil fertility and thus to partially replace conventional mineral nutrition.