<p>This study investigated the biohydrometallurgical recovery of rare earth elements (REEs) from coal fly ash (CFA) utilizing the mixotrophic bacterium <i>Alicyclobacillus ferrooxydans</i>. Two experimental series were conducted to optimize the extraction process. The first series examined the influence of pulp density variations (5%, 10%, and 15%), while the second explored the effect of pyrite addition (3&#xa0;g/L, 6&#xa0;g/L, and 9&#xa0;g/L) as an alternative to ferrous sulfate (FeSO₄·7H₂O). Both experimental series were performed over a 7-day bioleaching period. Post-bioleaching residues were analyzed using Fourier-transform infrared spectroscopy (FTIR) and X-ray diffraction (XRD) techniques. Results indicated that a 5% pulp density yielded the highest extraction levels of both heavy and light REEs compared to 10% and 15% pulp densities. Additionally, the introduction of pyrite demonstrated a tendency to enhance REE extraction levels. FTIR and XRD analyses corroborated the successful extraction of REEs by <i>A. ferrooxydans</i>. These findings emphasized the potential of biohydrometallurgical processes as a sustainable approach to recycling coal combustion by-products. By optimizing pulp density and utilizing pyrite as an alternative iron source, this method presents a promising solution for the efficient recovery of valuable REEs from CFA. This approach contributes to the circular economy and mitigates the environmental impact associated with traditional REE extraction methods. The optimization of pulp density and the use of pyrite as an iron source represent novel contributions to the field of biohydrometallurgical REE recovery. Further research is warranted to explore the scalability and economic viability of this process for industrial applications.</p> Graphical Abstract <p></p>