The health risks associated with manganese (Mn) in drinking water, and an improved understanding of Mn accumulation within, and subsequent release from, distribution systems, have increased the need for robust, sustainable treatment options to minimize Mn concentrations in finished water. Biofiltration is an established and effective method to remove Mn in groundwater however, Mn removal in surface water biofilters is an emerging treatment process that has not been extensively studied. Seasonal variations in water temperature can present an operational challenge for surface water biofilters which may see reduced Mn removal under colder conditions. This study examined the microbiomes of surface water biofilters at three utilities (ACWD WTP, WTP B, and WTP D) which all experienced similar seasonal fluctuations in influent water temperature. High Mn removal was observed at the ACWD WTP for much of the year, but Mn removal decreased with a concurrent decrease in the influent water temperature (58% ± 22%). In contrast, both WTP B and WTP D achieved year-round Mn removal (84% ± 5% and 93% ± 8% respectively). Marker gene (16S rRNA) sequencing analysis of the biofilter microbiomes identified a high abundance of Betaproteobacteria in WTP B and WTP D (37% ± 12% and 21% ± 3% respectively), but a low abundance of Betaproteobacteria in the ACWD WTP (2% ± 2%). The microbiomes of new bench-scale biofilters, in operation at the ACWD WTP, were also investigated. The abundance of Betaproteobacteria was significantly greater (p < 0.05) after the biofilters had acclimated than before acclimation, and differential abundance analysis identified 6 genera within the Betaproteobacteria class were enriched in the acclimated microbiome. Additionally, the acclimated biofilters were able to maintain high Mn removal performance (87% ± 10%) when the influent water temperature decreased to 10 °C or less. Further analysis of previously published studies found the abundance of Betaproteobacteria was also significantly greater (p < 0.001) in biofilters with sustained Mn removal than in biofilters which did not treat for Mn as a contaminant, despite differences in design scale, source water, and media type. Microbiome network analysis identified multiple co-occurrence relationships between Betaproteobacteria and Mn oxidizing bacteria in the WTP B and WTP D biofilters, suggesting indirect contributions by Betaproteobacteria to biological Mn oxidation. These co-occurrence relationships were not present in the full-scale ACWD WTP microbiome. Whether the role of Betaproteobacteria in biological Mn oxidation is direct, indirect, or a combination of both, they are consistently present at a high abundance in both groundwater and surface water biofilters with sustained Mn removal, and their absence may contribute to the seasonal fluctuations in Mn removal observed at the ACWD WTP. This new insight to Betaproteobacteria and their role in Mn biofiltration could contribute to water innovation and design that would improve the reliability of Mn removal.