This research aims to optimize the growth of local microorganisms from rumen fluid waste cultured in medium with different C/N ratios and to evaluate ammonia removal from chicken manure using local microorganisms as starters. Rumen fluid waste, molasses, and tofu dregs were used in this study. Local microorganisms (LMOs) production was carried out by using three treatments of cultured growth medium with different carbon-to-nitrogen (C/N) sources formulated by molasses:tofu dregs liquid that is P1 (1:1), P2 (1:2), and P3 (1:3). Then, all treatment media were compared to a control P0 (natural rumen fluids). The results showed that P1, P2, and P3 had various initial C/N ratios of 25.30±1.00, 24.46±0.84, and 23.94±0.94, respectively. The local microorganisms product was represented by colonies through total plate count: P1 (4.17±0.60)×10⁸ CFU/mL; P2 (3.58±0.06)×10⁸ CFU/mL; P3 (0.41±0.24)×10⁸ CFU/mL, and total lactic acid bacteria: P1 (1.37±0.40)×10⁸ CFU/mL; P2 (2.90±0.40)×10⁸ CFU/mL; P3 (0.41±0.26)×10⁸ CFU/mL, whereas total coliforms were not detected. Next, the LMO exhibited proteolytic activity, as indicated by a clear zone in the growth of the plate media containing 1% skim milk. Moreover, the application of LMO to evaluate its ability was inoculated to the layer chicken manure, resulting in a significant (P<0.05) decrease in ammonia levels compared to the control (6463.52 ppm). Treatment P1 decreased the ammonia level at 4165.32 ppm, which indicated removing about 35.5% of the ammonia concentration. Finally, we concluded that preliminary laboratory results indicate potential local microorganisms from rumen fluid that have been cultured with a 1:1 carbon-to-nitrogen source provide optimal conditions for microorganisms growth and can be used to reduce the ammonia in chicken manure significantly; therefore they can be used as a potential bioactivators for livestock waste processing.