The objective of the experiment was to determine the effects of different levels of phytase (0, 150, 250, and 350 phytase units (FTU)/kg, F) produced by Aspergillus niger in the diets of hens fed decreased contents of non-phytate phosphorus (1.8 and 2.1 g/kg, NPP) on the hen performance, egg quality, digestibility of calcium and phosphorus in the ileum, and representation of bacterial species in the ileum and caecum. The diet with 2.1 g/kg of NPP and 350 FTU/kg significantly decreased egg weight (P < 0.001) and egg mass production (P < 0.001). However, this treatment had the highest values for shell percentage (P = 0.002), shell thickness (P = 0.006), and shell index (P = 0.003). The supplementation with F at 350 FTU/kg to the diet with 1.8 g/kg of NPP increased the shell quality to a level that was comparable with the eggs from the hens fed the diet with only 2.1 g/kg of NPP. With the addition of F (350 FTU/kg) to the mixed feed with 1.8 g/kg of NPP, the digestibility of calcium and phosphorus in the ileum increased by 6.1% and 7.4%, respectively, although the increases were not significant. Additionally, the frequency of Lactobacillus spp. was higher in the ileum and caecum of hens fed the diet enriched with F than in those fed a diet without F addition. The dietary manipulations with NPP and F improved some performance and shell quality characteristics, and the addition of 3-phytase at a level of 350 FTU per kg to the low-P diet increased the digestibility of minerals and changed the microflora of the digestive tract.