Cellular damage resulting from elevated levels of free radicals can lead to persistent health issues. <i>Pleurotus floridanus</i>, an edible white oyster mushroom, is rich in β-glucans with potent antioxidant and anti-inflammatory properties. In this research, we examined the β-glucan content, total phenolic content, as well as antioxidant and anti-inflammatory potential of hot water extracts with varying particle sizes (< 75, 75-154, 154-300, and 300-600 μm) of both whole and sliced fruiting bodies of <i>P. floridanus</i>. The findings revealed that the в-glucan content increased as the particle size increased, although no significant differences were observed. Conversely, smaller particle sizes (< 75 μm) of whole and sliced fruiting bodies of <i>P. floridanus</i> exhibited higher phenolic content, 2,2-diphenyl-1-picryl-hy-drazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) scavenging activity, and reducing ability compared with larger particle size (> 75 μm). Of the four samples (AW2, AW3, AS1, and AS2) with the highest antioxidant activity selected for anti-inflammatory assays, all demonstrated the ability to reduce nitric oxide and tumor necrosis factor-alpha levels, but did not enhance interleukin-10 expression in lipopolysaccharide-stimulated RAW264.7 cells. Interestingly, particle size < 75 to 300 μm did not appear to influence the anti-inflammatory activity, because no significant differences were observed among the particle sizes. Therefore, a particle size < 300 μm in a <i>P. floridanus</i> hot water extract could serve as a valuable source of antioxidant and anti-inflammatory compounds to counteract the harmful effects of free radicals.