To assess the feasibility of the Anammox process as a cost-effective post-treatment step for anaerobic sewage treatment, the simultaneous effects of organic carbon source, chemical oxygen demand (COD)/N ratio, and temperature on autotrophic nitrogen removal was studied. In batch experiments, three operating conditions were evaluated at 14, 22 and 30 °C, and at COD/N ratios of 2 and 6. For each operating condition, containing 32 ± 2 mg NH4+-N/L and 25 ± 2 mg NO2−-N/L, three different substrate combinations were tested to simulate the presence of readily biodegradable and slowly biodegradable organic matter (RBCOD and SBCOD, respectively): (i) acetate (RBCOD); (ii) starch (SBCOD); and (iii) acetate + starch. The observed stoichiometric NO2−-N/NH4+-N conversion ratios were in the range of 1.19–1.43, and the single or simultaneous presence of acetate and starch did not affect the Anammox metabolism. High Anammox nitrogen removal was observed at 22 °C (77–84%) and 30 °C (73–79%), whereas there was no nitrogen removal at 14 °C; the Anammox activity was strongly influenced by temperature, in spite of the COD source and COD/N ratios applied. These results suggest that the Anammox process could be applied as a nitrogen removal post-treatment for anaerobic sewage systems in warm climates.