Agaricus bisporus (white button mushroom) is one of the most popular culinary-medicinal mushrooms worldwide. This species has for decades been the subject of numerous scientific studies. The aim of this study was to examine the pro- or anti-inflammatory properties of A. bisporus and biomass extracts from in vitro cultures growing in Oddoux medium enriched with α-linolenic acid in colon epithelial Caco-2 cells activated with lipopolysaccharide (LPS) and tumor necrosis factor (TNF)-α. Incubation of Caco-2 cells with A. bisporus extracts resulted in decreased expression of cyclooxygenase-2 and prostaglandin F2α receptor compared with the LPS- and/or TNF-α-activated cells, whereas the expression of nuclear factor (erythroid-derived 2)-like 2 increased after incubation. Interleukin-6 level decreased significantly in Caco-2 cells after supplementation with mushroom extracts. The amounts of monoun-saturated and polyunsaturated fatty acids differed significantly in Caco-2 cell membranes after supplementation with A. bisporus extracts. Our findings suggest the presence of anti-inflammatory and antioxidant properties of A. bisporus biomass extracts from in vitro cultures.