Routine identification of pathogenic microorganisms predominantly based on nutritional and biochemical tests is a time-consuming process; the delay may lead to fatal consequences at times. In this work, nanoparticle-induced nanoSPR enhanced IR spectroscopy was used in conjunction with a background elimination data processing algorithm to directly identify microorganisms in mixed cultures. It was demonstrated that the microbial composition of mixtures of different E. coli strains could be identified with 100% accuracy. The procedure was also applied to determine the presence or absence of pathogenic microorganisms in a simple but real food matrix (apple juice). Results indicated that microorganisms in a cocktail of up to eight different species suspended in an apple juice matrix could be identified for its presence or absence with 100% accuracy.