The inhalation of natural nanoparticles (NPs) emitted from volcanic activity may be a risk to human health. However, the literature rarely reports the fate and response of NPs once in contact with lung fluids. In this work, we studied the particle size distribution of ashfall from Popocatépetl volcano, Mexico. The collected ashes (n = 5) were analyzed with scanning electron microscopy (SEM) to obtain the elemental composition and morphology, and to determine the size of the ash particles using ParticleMetric software (PMS). The PMS reported most of the ash to have submicrometric size (<1 μm) and an average equivalent circle of 2.72 μm. Moreover, to our knowledge, this study investigated for the first time the behavior of ash NPs at different times (0 to 24 h) while in contact with in vitro lung fluid, Gamble Solution (GS) and Artificial Lysosomal Fluid (ALF) using dynamic light scattering (DLS). We found a large variability in the hydrodynamic diameter, with values less than 1 nm and greater than 5 μm. Furthermore, aggregation and disaggregation processes were recognized in GS and ALF, respectively. The results of this study increase the knowledge of the interaction between NPs and lung fluids, particularly within the alveolar macrophage region.