Currently, human populations are continuously exposed to numerous environmental xenobiotics, including a broad range of pesticides such as glyphosate (Glyph) and its formulations (glyphosate-based herbicides, GBH), the most widely used worldwide. Increasing evidence indicates that the central nervous system is a target of both Glyph and GBH toxicity; however, the underlying mechanisms remain poorly understood. Studies from our group have reported behavioral impairments, including deficits in recognition and spatial memory in exposed rats, as well as marked effects on hippocampal neurons, impacting morphology, neuronal maturation, and synapse formation. The aim of this study was to determine whether GBH exposure induces alterations in young neuronal cultures (24 and 48 h) and in mature neurons (21 DIV). Our results showed that in immature neurons, 0.5 mM Glyph-equivalent GBH delayed axonal and dendritic development, resembling the effects of pure Glyph at doses 50 times higher. In mature cultures, 0.05–0.1 mM Glyph-equivalent GBH reduced dendritic tree complexity and affected both the number and type of dendritic spines, comparable to effects caused by pure Glyph at doses 60 times higher. Moreover, preliminary assays from the hippocampus of treated rats showed a decrease in the activity of effectors of non-canonical Wnt pathways as well as in the expression of synaptic markers. These findings suggest that the presence of adjuvants in GBH enhances glyphosate neurotoxicity.