This study investigates the potential synergy effect of Ganoderma lucidum and Lentinus edodes mushroom extracts delivered through niosomes (composed of Span 40, Tween 40, and cholesterol) for anti-cancer and antimicrobial applications. GC–MS analysis revealed bioactive compounds such as d-limonene, ascorbic acid, and ergosterol in the extracts, known for their anti-cancer and antimicrobial effects. The niosomal formulations exhibited improved physical properties, with sizes ranging from 96 to 160 nm and polydispersity index (PDI) values in the range of 0.29–0.54, suggesting that the niosomes had an acceptable size distribution and homogeneity. In cell culture studies, the niosomal formulations demonstrated significant cytotoxic effects on A549 lung cancer cells, with the combination of both extracts leading to a notable decrease in cell viability to 14.86%. Additionally, the combination of G. lucidum and L. edodes extracts in niosomal form showed varying and potent antimicrobial properties against pathogenic bacteria (Pseudomonas aeruginosa and Staphylococcus aureus) and fungi (Candida albicans) with lower minimum inhibitory concentration (MIC) values compared to their free form, indicating a potential synergistic effect. The findings highlight the potential synergistic anti-cancer and antimicrobial effects of the combined mushroom extracts delivered through niosomes, offering valuable insights for developing novel drug delivery systems.