As pre-treatment processes improve biogas production during anaerobic digestion of sewage sludge, it is expected that the microbial communities of digesters will adapt to the exposition to pre-treated substrate in terms of their composition or activity. In this study, the influence of sono-thermal pre-treatment over the microbial communities of digesters operated at decreasing solids retention times (SRT) was assessed. Differences in the microbiome and specific activities were observed and associated with pre-treatment and SRT. Pre-treatment increased methane yields (19.1–29.9%), bacterial range-weighted richness (27.8–46.5%) and archaea concentration (one order of magnitude in terms of 16S rDNA gVS−1), without causing process inhibition or instability. No effect over archaea richness was observed, which was dominated by the presence of acetoclastic methanogens. Hydrolytic and acidogenic activities were similar among the reactors and across SRT, with values of 1.55–2.28 gCOD−1d−1 and 1.45–1.88 gCOD−1d−1, respectively. Methanogenic activity showed a positive correlation with SRT, with values of 0.12–0.13 gCOD gVS−1 d−1 at 7.5 days SRT and 0.31–0.37 gCOD gVS−1 d−1 at 30 days SRT. Overall, the results shown that the main drivers of the observed effects over methane yield and the microbiome were organic loading rate and the solubilization caused by pre-treatment.