Bacterial sensing based on quantum cascade laser spectroscopy coupled with diamond or gallium arsenide thin-film waveguides is a novel analytical tool for gaining high-resolution infrared spectroscopic information of planktonic and sessile bacteria, as shown in the present study for Escherichia coli. During observation periods of up to 24 h, diamond and gallium arsenide thin-film waveguide laser spectroscopy was compared to information obtained via conventional Fourier transform infrared spectroscopy. The proliferation behavior of E. coli at those surfaces was complementarily investigated using atomic force microscopy and scanning electron microscopy.