The aim of this study was to evaluate the short‐term effect of probiotic inoculation on the abundance of heterotrophic and ammonia‐oxidizing bacteria in mature biofloc, as well as on total suspended solids (TSS), chlorophyll‐a and nitrogenous compounds in water. A completely randomized design consisting of five treatments (three commercial probiotics, one native consortia and one control) was performed. At the beginning of the experiment (day 1), each treatment was inoculated with the respective probiotic: PondPlus® (PP), Efinol® PT (EF) and Epicin® ponds (EP), native consortia UE, whereas the control was not inoculated. Water parameters and bacterial abundance were evaluated at 0, 2, 4, 6 and 8 days. The addition of probiotics, either native or commercial, did not show any significant effect on the TSS, Chl‐a and colony‐forming unit (CFU) of heterotrophic bacteria when they were added to the systems containing mature biofloc. A significant increase in ammonium oxidizing bacteria was registered with the probiotics PP and EP, although the levels of total ammonia nitrogen, NO3‐N and NO2‐N were statistically similar among all treatments. Modifications on most of the parameters measured were associated with the factor of time, rather than the inclusion of probiotics. Results suggest that the bacterial conglomerates in mature stage contain well‐established bacterial communities that are difficult to be affected by the addition of probiotics.