Recently, mushroom polysaccharides have been explored to attribute to vital biologically important functions, and several extraction techniques can be employed, therefore, polysaccharides were extracted from the edible mushroom Calocybe indica to explore its functionality. Multiple enzymes viz., cellulase, pectinase, and protease (1:1:1) at temperature 47 °C and pH 4.64 with an extraction time of 2 h yielded 7.24 % polysaccharide content. The thermograph curve of polysaccharides showed two-stage decomposition at a different temperature range and decomposition of polysaccharides initiated with an onset temperature of 226.77 °C and a maximum peak at 248.90 °C. Hydrodistillation processed Eucalyptus globulus leaf oil was characterized using the chromatography technique and eucalyptol, p-cymene, Γ-terpinene, 4-epi-cubebol, spathulenol, viridiflorol, and p-mentha-1,5-dien-8-ol was observed as major components. As well, we formulated nanoemulsion using mushroom polysaccharide and eucalyptus leaf oil with 140.8 nm and evaluated synergistic antimicrobial and antibiofilm activity. MIC and MBC values for Pseudomonas aeruginosa, E. coli, and S. typhi were 12.50–3.12 and 6.25–1.56, and for S. aureus were 6.25, 6.25, 3.12, and 3.12, 3.12, 1.56 and for C. albicans the values were 12.50, 12.50, 6.250 and 6.25, 6.25, and 3.12 μL/mL respectively. The polysaccharides, essential oil, and nanoemulsion showed remarkable antibiofilm activity against S. aureus with inhibition of 57.42 ± 0.19, 59.62 ± 0.15, and 69.34 ± 0.19 %, while E. coli showed the least antibiofilm activity. However, all three tested samples showed significant (p < 0.05) differences against tested pathogenic microorganisms with inhibition of biofilm formation. Therefore, it could be inferred that the synergistic properties of essential oils with mushroom polysaccharides are a promising strategy to enhance antimicrobial efficacy and control foodborne pathogens.