Diversity of technological conditions and of raw materials from which biochar is produced is the reason why its soil application may have a varied effect on chemical and biological properties of soil and soil ecotoxicity. The aim of this study was to evaluate the effect of the addition of poultry litter (5.00tDMha−1) and biochar obtained from this material in doses of 2.25t and 5tDMha−1 on soil enzymatic activity, soil ecotoxicity and grass crop yield (pasture grass mix). The research was carried out under field conditions. No significant effect of biochar amendment on soil enzymatic activity was observed. The biochar-amended soil was low-toxic to Vibrio fischeri and non-toxic to Heterocypris incongruens. Application of poultry litter biochar in doses of 2.25t and 5tDMha−1 contributed to an increase in plant biomass production by 32% and 30%, respectively compared to the control (C). Biochar had more adverse effect on soil enzymatic activity and grass crop yield than non-converted poultry litter, but it significantly reduced soil toxicity to H. incongruens and V. fischeri.