The chemical mechanisms by which biochar addition affected soil microorganisms have been extensively studied. However, few studies investigated the effect of physical alteration induced by biochar application on microorganisms in soils. The study focused on how the pore structure affected microbial diversity and community structure in an Ultisol amended with straw-derived biochars. The nitrogen adsorption isotherm (NAI) and mercury intrusion porosimetry (MIP) were used to measure the soil pore characteristics. The bacterial and fungal community composition and diversity were analyzed by the sequencing of V4-V5 of 16 S rRNA gene and ITS1 gene, respectively. MIP results showed that biochar increased the total porosity, total pore volume, average pore diameter and the volumes of > 75, 30–75 and 5–30 µm pores in soils. The straw feedstock and pyrolysis temperature of biochar affected the microbial diversity and community structure in soils. The soil amended with RB550 had the highest Shannon diversity of bacteria and fungi, while the soil treated with CB350 had the highest bacterial abundance. The addition of biochar mainly increased the relative abundances of bacterial genera Actinospica, Ellin6067, Streptomyces and Massilia, while decreased the abundance of Pseudomonas, Methylobacterium and Nitrosospira. However, the fungal genera had a greater variation in biochar-amended soils. The > 5 µm pores in soils had positive effects on the microbial diversity and abundance. The bacterial genera that were acidophilic and aerobic had positive correlations with the volumes of > 75, 30–75 and 5–30 µm pores, especially Ellin6067, Flavisolibacter and Haliangium. Inversely, the genera that were facultative anaerobic (Methylobacterium, Pseudomonas and Nitrosospira) and anaerobic (Christensenellaceae_R-7_group) showed a positive correlation with the volume of < 5 µm pores or no obvious regularity. Most fungal genera tended to live in the larger pores of > 5 µm and could extend into smaller pores. Therefore, the pore characteristics largely determined the microbial community structure in the biochar-amended soils.