This paper focuses on evaluation of in vitro antimicrobial activity of two natural antibiotics, monensin (1) and lasalocid (2) and their metal complexes with Tl(I) (3 and 4), La(III) (5) and Gd(III) (6) against Mycobacterium tuberculosis, Saccharomyces cerevisiae and Fusarium oxysporum f.sp albedinis using POM analyses. Results indicated that monensin (1) and its Tl(I) complex (3) show remarkable bioactivity with MIC values of 0.15-0.34 M and 0.1-0.23 M against Fusarium oxysporum f.sp albedinis and Saccharomyces cerevisiae yeast, respectively. Preliminary screening results of selected ionophores exhibited potent antimicrobial activity; these ionophores could be considered as good potential drug candidates as antifungal agents against various fusarioses and also as potential antitubercular agents for future discovery of new drug design.