Anaerobic digestion (AD) is a suitable process to use manure for biogas production. During this process, antimicrobial resistant (AMR) bacteria are reduced. In this study, we investigated the reduction of extended-spectrum beta-lactamase (ESBL)-producing, fluoroquinolone-resistant, and total Escherichia (E.) coli concentrations in chicken manure during AD. AD was performed at 30 °C and 37 °C (mesophilic temperature range), with and without sawdust addition as a lignocellulosic biomass. The initial concentrations of total E. coli were 6.43 log10CFU/g to 7.84 log10CFU/g. The concentration of Fluoroquinolone-resistant E. coli was approx. 6.05 log10CFU/g (6.70%), and of ESBL-producing E. coli approx. 5.48 log10CFU/g (0.99%). During AD, we observed that temperature had the main influence on the bacterial reduction, as E. coli abundance was below the detection limit after day 7 at 37 °C and after day 14 at 30 °C. At 37 °C we observed higher amounts of free ammonia, which is an inhibitor of the AD process. The carbon-to-nitrogen (C/N) ratio has an important impact on the AD process because a higher C/N ratio decreases the amount of generated total ammonia nitrogen. However, we did not observe a significant difference in AMR and total E. coli reduction between chicken manure with a natural C/N ratio (10:1) and an increased C/N ratio (25:1).