Staphylococcus aureus is one of the main pathogenic microorganisms found in milk and dairy products and has been involved in bacterial foodborne outbreaks in the past. Current enumeration techniques for bacteria are very time-consuming, typically taking 24 h or longer, and bacterial antagonism in the form of lactic acid bacteria (LAB) may inhibit the growth of S. aureus . Therefore, the aim of this investigation was to establish the accuracy and sensitivity of rapid nondestructive metabolic fingerprinting techniques, such as Fourier transform infrared (FT-IR) spectroscopy and Raman spectroscopy (RS), in combination with multivariate analysis techniques, for the detection and enumeration of S. aureus in milk, as well as to study the growth interaction between S. aureus and Lactococcus lactis ssp. cremoris , a common LAB. The two bacterial species were investigated both in a pure monoculture and in a combined inoculated coculture after inoculation into ultraheated milk during the first 24 h of growth at 37 °C. Plating techniques were used to obtain primary reference data for viable bacteria counts. Principal component discriminant function analysis, canonical correlation analysis, partial least-squares (PLS), and kernel PLS (KPLS) multivariate statistical techniques were employed to analyze the data. FT-IR provided very reasonable quantification results both with PLS and KPLS, the latter providing marginally better predictions, with correlation coefficients in the test set (Q(2)) and training set (R(2)) varying from 0.64 to 0.76 and from 0.78 to 0.88 for different bacterial sample combinations. RS results were less encouraging with high degrees of error and poor correlation to viable bacterial counts. S. aureus growth was not inhibited by the presence of the LAB, but metabolic fingerprinting of the coculture indicated that the phenotype of this dual bacterial culture was closer to that of pure LAB cultures. In conclusion, FT-IR spectroscopy in combination with the above multivariate techniques appears to be a promising discrimination and enumeration analytical technique for the two bacterial species. In addition, it has been demonstrated that the L. cremoris metabolic effect in milk dominates that of S. aureus even though there was no growth antagonism observed.