To evaluate the anticancer, antioxidant and antimicrobial activities along with total phenolic and flavonoids contents extracted from Lespedeza bicolor indigenous to Khyber Pukhtoonkhwa, Pakistan.Anticancer activity against human lung carcinoma (LU-1) and human prostate carcinoma (LnCap), antimicrobial against Aspergillus fumigates, Aspergillus niger, Fusarium solani, Mucor sp Klebsiella pneumonia and Enterococcus and antioxidant activity on 1,1-Diphenyl-2-picryl-hydrazyl (DPPH) and Hydrogen peroxide radicals scavenging activity were assessed in methanol extract of Lespedeza bicolor.Lespedeza bicolor aerial parts and root extract showed IC50 value of 12.5 and 50 μg/mL against LU-1 whereas, ≤ 12.5 and 12 ug/mL were calculated against LnCap cell line. Minimum inhibition concentration (MIC) value of 20-35 ug/mL has been observed against Aspergillus fumigates, Aspergillus niger, Fusarium solani and Mucor sp in comparision with 1-2.5 ug/mL of Terbinafine used as a standard fungicide. MIC value of 20 and 35 ug/mL of Lespedeza bicolor aerial parts and root extract against bacterial pathogen Klebsiella pneumonia and 20-50 ug/mL against Enterococcus has been measured. DPPH radical scavenging activity of Lespedeza bicolor with IC50 values of ≤ 50 ug/mL and ≤ 200 ug/mL was observed whereas, hydrogen peroxide scavenging activity with IC50 values of ≤ 25 ug/mL for aerial parts and ≤50 ug/mL for the root extract of Lespedeza bicolor has been shown.These data suggested that the methanolic extract of Lespedeza bicolor could be potential candidates for natural antioxidants and anticancer.