The production of acid mine drainage (AMD) involves oxidation of FeS2 to SO42−, during which a variety of intermediate sulfur forms (S2O32−, S0, SnO62−, SO32−) are generated. This study aimed to characterize the spatiotemporal distributions of different forms of these intermediates and their relationships to environmental factors, focusing on an abandoned pyrite mine area. Samples were collected from different water stages and the physicochemical factors were determined on site. High performance liquid chromatography, ion chromatography, and Illumina high-throughput sequencing were used to determine the distributions of iron and sulfur forms and the microbial community structure at each site. Pearson and Spearman correlation were used to analyze the relationships between the distributions of different forms of sulfur and environmental factors during the formation and migration of AMD. The results suggested that SO42− mainly originated from gypsum dissolution and oxidation of the coal mine and FeS2. The dry season was associated with lower water pH and temperature and higher DO and ORP. The maximum correlation coefficient between TFe and SO42− decay was 0.9308, which could be attributed to the formation of sulfate secondary iron-containing minerals. SO42− pollution decreased with increasing migration distance of AMD and showed seasonal variation closely related to precipitation and groundwater flow. The abundance and diversity of microbial community decreased with the production of AMD, mainly acidophilus and sulfur/iron-oxidizing bacteria. Ferrovum occupied an absolute dominant position in weakly acidic samples, and Acidibacter and Sphingomonas were not polluted. Neutral samples include Lachnospiraceae NK4A136 group, Ralstonia, Sinomonas, etc. pH and SO42− showed negative correlations with DO, temperature, and ORP, whereas the dominant strain Acidithiobacillus was positively correlated with SO42−. Increases in water temperature and ORP promoted the transformation of sulfur. The regulation of sulfur conversion to acid is key for developing strategies for preventing and reversing AMD pollution.