Context. The term macroturbulent broadening is commonly used to refer to a certain type of non-rotational broadening affecting the spectral line profiles of O- and B-type stars. It has been proposed to be a spectroscopic signature of the presence of stellar oscillations; however, we still lack a definitive confirmation of this hypothesis. Aims: We aim to provide new empirical clues about macroturbulent spectral line broadening in O- and B-type stars to evaluate its physical origin. Methods: We used high-resolution spectra of 430 stars with spectral types in the range O4 - B9 (all luminosity classes) compiled in the framework of the IACOB project. We characterized the line broadening of adequate diagnostic metal lines using a combined Fourier transform and goodness-of-fit technique. We performed a quantitative spectroscopic analysis of the whole sample using automatic tools coupled with a huge grid of fastwind models to determine their effective temperatures and gravities. We also incorporated quantitative information about line asymmetries into our observational description of the characteristics of the line profiles, and performed a comparison of the shape and type of line-profile variability found in a small sample of O stars and B supergiants with still undefined pulsational properties and B main-sequence stars with variable line profiles owing to a well-identified type of stellar oscillations or to the presence of spots in the stellar surface. Results: We present a homogeneous and statistically significant overview of the (single snapshot) line-broadening properties of stars in the whole O and B star domain. We find empirical evidence of the existence of various types of non-rotational broadening agents acting in the realm of massive stars. Even though all these additional sources of line-broadening could be quoted and quantified as a macroturbulent broadening from a practical point of view, their physical origin can be different. Contrarily to the early- to late-B dwarfs and giants, which present a mixture of cases in terms of line-profile shape and variability, the whole O-type and B supergiant domain (or, roughly speaking, stars with MZAMS ≳ 15 M⊙) is fully dominated by stars with a remarkable non-rotational broadening component and very similar profiles (including type of variability). We provide some examples illustrating how this observational dataset can be used to evaluate scenarios aimed at explaining the existence of sources of non-rotational broadening in massive stars. Full Table 1 is only available at the CDS via anonymous ftp to http://cdsarc.u-strasbg.fr (http://130.79.128.5) or via http://cdsarc.u-strasbg.fr/viz-bin/qcat?J/A+A/597/A22