To clarify the effects and mechanisms of biochar combined with acid-tolerant Bacillus subtilis on red soil acidification amelioration and biodiversity improvement, this study conducted an incubation experiment to investigate the dynamic changes and interrelationships of soil pH, nutrient content, and microbial diversity after co-application of maize straw biochar and acid-tolerant Bacillus subtilis Bs-m9. The results showed that the combined treatment (biochar + Bs-m9) significantly increased soil pH by 0.37 units, reduced exchangeable acidity and exchangeable aluminum by 24.6% and 9.6%, respectively, and increased organic matter by 64.0% within a short term (day 6). Under long-term treatment (day 60), exchangeable aluminum further decreased by 19.8% compared to the control, while organic matter and available phosphorus exhibited stable enhancement. Microbial community dynamics analysis revealed that short-term treatment temporarily reduced bacterial richness and Shannon index compared to the control, but these metrics significantly reversed after long-term treatment (60 days), increasing by 11.62% and 3.60%, respectively. This reversal of bacterial diversity decline drove the directional succession of oligotrophic phyla, including Acidobacteriota and Chloroflexi, with specific enrichment of the WPS-2 functional group (15.68% and 67.2% higher than the control on days 6 and 60, respectively) and Basidiomycota (55.2% and 207.1% higher than the control on days 6 and 60, respectively). Redundancy analysis indicated that bacterial communities under the combined treatment were initially driven by organic matter (R2 = 0.46) and total phosphorus (R2 = 0.61) on day 6, but shifted to exchangeable aluminum dominance (R2 = 0.79) by day 60. Fungal diversity remained regulated by dissolved organic carbon throughout incubation (R2 = 0.81-0.45). In conclusion, the combined application of biochar and acid-tolerant Bacillus subtilis mitigates red soil acidification in short time, enhances microbial diversity, optimizes community structure, and demonstrates promising potential for application in red soil remediation.