This study evaluated startup strategies for mainstream polishing anammox moving bed biofilm reactors (MBBRs) without anammox bacterial (AMX) biomass inoculation. Two types of startups were tested: anammox only (no external carbon addition) and partial denitrification/anammox (PdNA) with glycerol addition. Reactors were started with either virgin carriers or carriers with a preliminary biofilm from a mainstream aerobic integrated fixed‐film activated sludge (IFAS) process. Three pilot‐scale startups were completed under the following conditions: anammox‐only with preliminary biofilm carriers, PdNA with preliminary biofilm carriers, and PdNA with virgin carriers. AMX presence was confirmed via quantitative polymerase chain reaction (qPCR) after 57, 57, and 77 days, respectively. Prior to AMX detection, average influent concentrations of ammonia and nitrite ranged from 1.7–2.7 mg/L and 0.98–1.8 mg/L, respectively. This study demonstrated that AMX can be grown on carriers without AMX seeding under mainstream conditions (temperature 17–29°C, low ammonia and nitrite), regardless of whether nitrite came from upstream or partial denitrification within the reactor. This study also showed that using preliminary biofilm carriers can decrease startup time by approximately 1 month. These results address critical questions for moving mainstream anammox processes to full‐scale implementation, and suggest that PdNA MBBRs are feasible and sustainable for full‐scale ammonia, nitrate, and nitrite polishing to meet stringent total nitrogen requirements. Practitioner Points This research will help utilities develop methods for starting up mainstream anammox MBBRs without the barrier of anammox biomass seeding. Preliminary biofilm carriers accelerated startup time in a PdNA MBBR, but a virgin carrier reactor started up in a similar timeframe, contrary to expectations. Also, contrary to expectations, high concentrations of ammonia and nitrite are not necessary for startup of an anammox or PdNA MBBR.