The present study describes the exploitation of microbial biodiversity from Western Ghats of Kerala for screening of bioactives having β-lactamase inhibitory activities. A total of 700 pure cultures were isolated and were screened for antibacterial activity against a β-lactam resistant Bacillus cereus strain (PL 10) isolated from the same niche. Bioactive extracts made from 45 isolates showed inhibitory activities against PL 10, of which two strains showed inhibition of extended spectrum β-lactamase (ESBL) producing Klebsiella ESBL1101 and three strains inhibited methicillin-resistant Staphylococcus aureus (MRSA) strain MRSA831. All these five strains showed wide spectrum antimicrobial activity against various fungi and bacteria. These five cultures were identified by 16S rRNA sequencing and biochemical tests and the preliminary characterizations of their bioactive extracts were carried out. This study suggests the potential of bioactives from two inhibitor-producer strains, NII 167 and NII 1054, for being developed as inhibitors against wide spectrum β-lactam resistant strains.