This work attempts to assess the antimicrobial potential of actinobacteria isolated from limestone mining sites which hitherto, is an under-explored niche for exploring novel bioactive metabolites. Actinobacteria were selectively isolated from Mawsmai, Meghalaya, India, a limestone mining area, using different pretreatment methods. Forty-seven isolates were obtained, which were identified based on their morphological, biochemical and chemotaxonomical characteristics. Streptomyces was the dominant cultivable genera which constituted 76% of the isolates cultivated. All the isolates were screened for antimicrobial activity against three Gram-negative viz. Escherichia coli, Pseudomonas aeruginosa and Klebsiella pneumoniae, and three Gram-positive bacteria viz. Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus and besides, two candidal species viz. Candida albicans and C. tropicalis. 19% of the total isolates showed antibacterial activity against at least one of the test bacterial strains used. The identity of the four bioactive isolates viz. LD-21, LD-29, LD-34 and LD-39 was confirmed as Streptomyces sp. on the basis of their 16S rDNA sequence and 16S rRNA secondary structure analysis. These isolates showed antibacterial activity against at least two Gram-positive bacteria and all the four harbored at least one of the three biosynthetic gene clusters viz. type-I and type-II polyketide synthases and non-ribosomal peptide synthetase which are related to synthesis of bioactive metabolites.