Western Ghats of India represents one of the global biodiversity hotspots. The present study was carried out to isolate and determine antimicrobial, cytotoxic and antioxidant activity of actinomycetes from soils of different regions of Western Ghats of Shi Karnataka, India. A total of 182 isolates were recovered from 57 soil samples collected at 13 different regions of Western Ghat region of Shivamogga district by serial dilution technique. Out of 182 isolates, 17 isolates were chos determine antimicrobial, cytotoxic and antioxidant activity. Solvent extracts of these isolates exhibited varied antimicrobial potency against Gram positive and Gram negative bacteria, yeasts and molds. Ethyl acetate was found to be best solvent for extraction of bioactive metabolites from most of isolates followed by butanol and hexane. Cytotoxic activity, as evaluated by brine shrimp lethality bioassay, revealed the potency of extracts to cause dose dependent mortality of brine shrimp larvae. Solvent extracts exhibited dose dependent antioxidant activity, as evaluated by DPPH, ABTS, Metal chelating and Ferric reducing assays. Western Ghat actinomycetes appear to be promising resources of bioactive agents and can be exploited industrially. Further studies on recovery of active constituents from solvent extracts and their bioactivity determinations are to be carried out. Abstract Article InformationWestern Ghats of India represents one of the global biodiversity hotspots. The present study was carried out to isolate and determine antimicrobial, cytotoxic and antioxidant activity of actinomycetes from soils of different regions of Western Ghats of Shivamogga district, Karnataka, India. A total of 182 isolates were recovered from 57 soil samples collected at 13 different regions of Western Ghat region of Shivamogga district by serial dilution-plating technique. Out of 182 isolates, 17 isolates were chosen for solvent extraction and to determine antimicrobial, cytotoxic and antioxidant activity. Solvent extracts of these isolates exhibited varied antimicrobial potency against Gram positive and Gram negative bacteria, und to be best solvent for extraction of bioactive metabolites from most of isolates followed by butanol and hexane. Cytotoxic activity, as evaluated by brine shrimp lethality bioassay, revealed the potency of extracts to cause dose brine shrimp larvae. Solvent extracts exhibited dose dependent antioxidant activity, as evaluated by DPPH, ABTS, Metal chelating and Ferric reducing assays. Western Ghat actinomycetes appear to be promising resources of bioactive agents ed industrially. Further studies on recovery of active constituents from solvent extracts and their bioactivity determinations are to be carried out.