Amaranth seeds are characterized by a very high nutritional value, which depends on various environmental factors and variety. For this plant, it is crucial to maintain the appropriate post-harvest quality of the seeds, particularly in terms of fungal infections. The aim of this study was to evaluate the impact of increasing NPK doses, row spacing, and variety on the biodiversity of fungi colonizing amaranth seeds.For this purpose, a 3-year field experiment (2016–2018) was carried out involving the cultivation of two varieties – ‘Rawa’ and ‘Aztek’ – under the soil and climatic conditions of southeastern Poland. The experiment demonstrated that most of the analyzed research factors (NPK fertilization, row spacing, and variety) had a significant impact on the health status of amaranth seeds. Increasing NPK fertilization led to higher seed colonization by Alternaria alternata. Row spacing affected both the total number of fungal colonies and the population of Aspergillus brasiliensis on the seeds.In this regard, amaranth cultivation was more favorable with wide row spacing than narrow row spacing. The variety significantly influenced seed germination after harvest, and the total number of fungal colonies on the seeds, particularly of Aspergillus brasiliensis and Alternaria alternata. The variety ‘Rawa’ was characterized by lower seed germination capacity and was more frequently populated by pathogenic fungi, which negatively affected seed quality compared to the variety ‘Aztek’. Considering the production of seeds free of post-harvest biological contamination in southeastern Poland, it is advisable to cultivate the variety ‘Aztek’ in a wide row spacing system