A combination of Fourier-transform infrared (FTIR) spectroscopy, multivariate analysis and conventional microbiological assays were utilized to characterize and differentiate membrane biofouling formed in the presence of antiscalants. Based on the FTIR spectra of biofouled reverse osmosis membranes obtained after incubating with antiscalants and H. aquamarina (as model microorganism), it was found that the biofouling intensity and composition was dependent on the type of antiscalants used. The growth of the bacterium was also highly affected by the type of antiscalants as shown by the colony forming unit (CFU) counts. By combining the techniques of principle component analysis (PCA) and FTIR, it was demonstrated that the biofouling was more intense and composed of proteins, polysaccharides and lipids, when polymer antiscalant was used. By applying PCA-FTIR with CFU counts, faster prediction of the effect of antiscalants on biofouling was made possible.