The high degree of microbial diseases and their multidrug resistant properties make the researchers to develop new class of antimicrobial agents. A modern and innovative approach of drug development is the use of metallic nanoparticles as new formulations of antimicrobial agents. In this study, microwave irradiated CaO nanoparticles (CaO-NPs) were used to determine their antimicrobial efficacy against gram negative and gram positive bacteria, as well as pathogenic yeast. The physiochemical properties of CaO-NPs were characterized by means of X-ray diffraction (XRD) and high resolution transmission electron microscopy (HRTEM). The nanoparticles consist of well dispersed agglomerates of grains with a narrow size distribution of 14-24 nm. The prepared CaO-NPs showed much higher antimicrobial activity against Pseudomonas aeruginosa (ATCC 27853) and Staphylococcus epidermidis (MTCC 435) in comparision to Candida tropicalis (NCIM 3110). The minimum inhibitory concentration (MIC) value of CaO-NPs was found within the range of 2-8 mM for all the above tested strains. This bioactive nanoparticle also inhibits the biofilm formation and may have future applications cheap and non toxic as antimicrobial drug for skin care product development.