Microbial pathogens are developing resistance against existing antibiotics, stressing the urgency for discovery of new therapeutic compounds. Actinomycetes, alone produce 70-80% of the available antibiotics. The chances of isolating undiscovered strains from the terrestrial habitats have diminished so that the search for novel products has switched to rare genera of actinomycetes from normal habitats or to discovery of strains/species found in unusual habitats. Rare genera of actinomycetes can be selectively isolated using various physical and chemical pre-treatment methods. Primary and secondary screening of isolates was done to determine their antimicrobial potential both qualitatively and quantitatively. Chemical structures of active metabolites were ascertained using chromatographic and spectrophotometric techniques. Since production of novel and more efficient antibiotics needs detection of high yielding bacteria, in the current study, we evaluated 5 soil samples (collected randomly from different zones of Jaipur (Rajasthan) viz. rhizosphere of plants, preserved green areas and forest soils) towards their antibiotic production potential using FTIR spectroscopy and HPLC methods. Based upon FTIR and HPLC analyses, the isolate A5 displayed promising results, inhibiting some important pathogenic bacteria and fungi.