Specifically designed samples have been analyzed to test the ability of Brillouin spectroscopy to provide reliable mechanical characterization of micro and nano-objects. The selected samples are polymeric films, whose transversal sizes from hundreds of nano- to some micro-meters cover the entire range of length-scales relevant in Brillouin scattering process. The experimental data highlight how, the size of the extended collective oscillation (acoustic phonons, in brief) is the lowest spatial resolution reachable in Brillouin mechanical characterization. Conversely, in the limit condition of phonon confinement, the technique provides the mechanical properties of nano-objects whose characteristic size is comparable with the phonon wavelength (⁓300 nm). Investigating acoustically heterogeneous materials, both size of heterogeneity and acoustic mismatch between adjacent regions are shown to be relevant in shaping the Brillouin response. In particular, a transition from a confined to a non-confined condition is obtained modulating the acoustic mismatch between the micro-objects and their local environment. The provided results and the derived analytic models for the data analysis will guide the interpretation of Brillouin spectra acquired in complex nano-structured samples such as cells, tissues or biomimetic materials. Our analysis can therefore generate new insights to tackle fundamental problems in mechanobiology or to characterize new bioengineered materials. Display omitted •Thin films of different traversals size are investigated as biomaterials models.•Brillouin spectroscopy measures the mechanical properties of micro and nano-objects.•Phonon confinement increases the spatial resolution of Brillouin spectroscopy.•Size and acoustic mismatch of heterogeneities alters Brillouin elastography.