SOLID state fermentation system (SSFS) efficiency depends on the proper selection of the microorganisms, substrates and environmental conditions. In this study, we investigated and optimized the antioxidant activity of the SSFS constructed using Pleurotus floridanus or Paecilomyces variotii on rice straw. The genotypic effect on antioxidant activity surpassed that of the environmental conditions. Peroxidase (POX) activity was expressed only in Pleurotus floridanus, but catalase (CAT) activity was expressed in both fungi, in favor of Paecilomyces variotii. The activities of both enzymes were maximized at low pH, low temperature and with ammonium chloride as the N source, but high moisture was optimal for POX and low moisture was optimal for CAT. Phenolic and flavonoid contents were higher in Paecilomyces variotii than Pleurotus floridanus, with preference for high moisture, low pH and urea in Paecilomyces variotii, and low moisture, low pH, and sodium nitrate in Pleurotus floridanus. The reducing power of Paecilomyces variotii was higher than that of Pleurotus floridanus, with positive moisture-dependence, low temperature, and gelatin preferences in Paecilomyces variotii, and negative moisture- and temperature-dependence and urea-preferences in Pleurotus floridanus. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) scavenging activity was higher in Paecilomyces variotii than Pleurotus floridanus, with negative dependence on moisture and temperature and limited nitrogen source effects in both fungi. Regarding the incubation period, maximal antioxidant activity was observed with a shorter incubation in the Paecilomyces variotii SSFS and a longer incubation in the Pleurotus floridanus SSFS.