Per- and polyfluoroalkyl substances (PFAS) are a diverse group of highly persistent contaminants that accumulate in terrestrial ecosystems and can exert adverse effects on non-target organisms. The earthworm Eisenia fetida is widely recognized as a sensitive bioindicator for assessing sub-lethal soil contamination. Previous studies have demonstrated that PFAS exposure induces oxidative stress, metabolic disruption, and reproductive impairment; by contrast, information on their neurotoxic effects remains limited, particularly for emerging PFAS and environmentally relevant mixtures. The present study investigated the effects of five PFAS—perfluorooctanoic acid (PFOA), perfluorobutanoic acid (PFBA), hexafluoropropylene oxide dimer acid (HFPO-DA, GenX), perfluoro(3-methoxybutanoic) acid (PFMOBA), and perfluoro(3-methoxypropanoic) acid (PFMOPRA)—on neurotransmitter systems in E. fetida. Earthworms were exposed to each compound individually and to an equimolar five-component mixture at concentrations of 0.06 and 229 µM. Neurochemical alterations were assessed on histological sections using a multiparametric immunohistochemical approach. Acetylcholinesterase (AChE) activity was evaluated using the Karnovsky staining method, whereas γ-aminobutyric acid (GABA) distribution was analysed by immunofluorescence. PFAS exposure significantly modulated neurotransmitter-associated responses within the circular muscle layer. Both AChE activity and GABA immunoreactivity differed from control values, indicating that PFAS engage specific neurochemical pathways beyond a generalized stress response. In particular, AChE staining intensity increased at the highest exposure concentration, especially in peripheral tissue regions, consistent with a localized increase in cholinergic (AChE) enzymatic activity within the muscle wall. GABA fluorescence also varied among treatments and anatomical regions, highlighting differential sensitivity of inhibitory neurotransmission to PFAS exposure. Preliminary observations further indicated detectable neurochemical alterations in organisms exposed to the PFAS mixture, supporting the sensitivity of this histological platform for identifying mixture-induced effects. These findings provide novel evidence of PFAS-mediated neurotoxicity in soil invertebrates and support the integration of neurochemical biomarkers into ecotoxicological assessments of both legacy and emerging PFAS.