This study evaluated the effectiveness of four biofilter substrates, Bioball (BB), dead coral (DC), zeolite (Ze), and activated carbon (AC), in supporting microbial colonization and improving water quality in a recirculating aquaculture system (RAS) for freshwater crayfish (Cherax quadricarinatus). A substrate-free control (Ct) was used for comparison over a 42-day culture period. Biofilter performance was assessed through bacterial abundance, water quality, and crayfish growth and survival. The substrate type strongly influenced nitrogen dynamics and microbial development. AC and Ze were most effective at reducing total ammonia nitrogen (TAN) and nitrite (NO2-), with AC lowering TAN from 0.21 mg/L to 0.11 mg/L and maintaining NO2- at 0.01–0.03 mg/L at 42 days of culture. These improvements corresponded to the highest survival rates of about 87% and 80% and to growth of about 1.12 and 1.05 g/day for AC and Ze, respectively. Bacterial counts were highest in AC and Ze, with AC supporting up to 21380 CFU/mL, 17783 CFU/mL, and 14380 CFU/mL of ammonifiers, Nitrosomonas, and Nitrobacter, respectively. Biochemical profiling revealed minimal diversity in Ct, with only Streptococcus sp., whereas BB, DC, and Ze supported complete nitrifying consortia. AC exhibited broad metabolic diversity, though Nitrosomonas detection was inconsistent between methods. The superior performance of AC was attributed to its high porosity and adsorption capacity, while Ze combined ion-exchange capability with microbial support. BB and DC performed moderately, whereas Ct was ineffective. Overall, AC and Ze emerged as the most promising substrates, demonstrating that indigenous microbial colonization is sufficient for stable nitrification and sustainable RAS management.