In this study, mycochemical screening was performed to estimate the in vitro biological activities and antimicrobial and antioxidant potential of Russula aeruginea and R. brevipes. Crude extracts of these mushroom species were obtained by using maceration techniques. Qualitative mycochemical analysis showed the presence of a variety of secondary metabolites such as terpenoids, alkaloids, flavonoids, reducing sugars, and anthraquinones. Antimicrobial activity of crude extracts was assessed by using agar well diffusion methods against virulent bacterial and fungal strains. Ethanolic extract of R. brevipes displayed maximum antibacterial activity, with a 28.4 ± 0.953 mm zone of inhibition against Bacillus subtilis. Likewise, ethanolic extract of R. aeruginea exhibited enhanced antifungal activity, with a 24.6 ± 0.333 mm zone of inhibition against Fusarium equiseti. The antioxidant potential of the ethanolic extracts was assessed using five assays: 2,2'-azino-bis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,2-diphenyl-1-picrylhydrazyl (DPPH), ferric reducing antioxidant power assay (FRAP), total flavonoid content (TFC), and total phenolic content (TPC). The maximum percent inhibition for R. aeruginea was 87.069 ± 0.007 mg/mL on ABTS radicals. In contrast, the maximum percent inhibition for R. brevipes was 42.166% ± 0.023% and 0.571 ± 0.005 mmol/L of FeSO4 using the DPPH and FRAP methods, respectively. TFC was the same for both mushrooms (0.113 ± 0.002 mg/100 g of catechin), and the maximum TPC was 0.066 ± 0.00 mg/100 g of gallic acid equivalents. From these results, it was concluded that R. aeruginea and R. brevipes could be a source of new antimicrobial and antioxidant agents and may be useful for the development of novel drugs in the future.