The spatiotemporal variation in meiofaunal assemblages were investigated for the first time in the Cabras Lagoon, the largest transitional system in the Sardinian Island (W-Mediterranean Sea). Two main environmental (salinity and trophic) gradients highlighted a significant separation of the three study sites across the lagoon, which were consistent through time. The environmental variability and habitat heterogeneity of the Cabras Lagoon influenced the meiofauna. In particular, salinity and dissolved oxygen, primarily, shaped the meiofaunal assemblage structure at the seaward site which was significantly different from both the riverine and the organically enriched sites. On the other hand, the trophic components (e.g., organic matter, Chlorophyll-a, and phaeopigments) and the different degrees of confinement and saprobity among sites were the secondary factors contributing mostly to the separation between the latter two sites. The lack of significant differences in the temporal comparison of the meiofaunal assemblage structure along with the very low contribution of temperature to the meiofaunal ordination indicated that this assemblage was more affected by spatial rather than by temporal variation. This pattern was also supported by significant differences between the three sites in several univariate measures, including total number of individuals, number of taxa, Pielou’s evenness, and the ratio between nematodes and copepods. Thus, the present study corroborates the hypothesis that meiofaunal organisms are good indicators of the spatial heterogeneity in transitional waters (TWs) and could have a greater species richness than that expected. Indeed, the Cabras Lagoon overall showed one of the highest meiofaunal richness values found from both Mediterranean and European TWs.