This study aims to investigate the trans-dentinal and direct cell viability of ammonia- and water-based silver fluoride treatments. Deep dentin discs were prepared, balanced for permeability and autoclaved. Three-dimensional cultures of odontoblast-like cells were transferred to the pulpal aspect of the dentin slices inside perfusion split chambers designed for dentin-barrier cytotoxicity test, following ISO 7405. An experimental resin-based glass ionomer cement and a polyvinylsiloxane impression material served as positive and negative controls, respectively. The experimental treatments included: (i) ammonia-based silver fluoride = SDF, (ii) SDF + potassium iodide = KI, (iii) water-based silver fluoride = SF, and (iv) SF + KI. Treatments were applied to the occlusal surface of dentin discs and cell viability (%) was assessed after 24 h using the methylthiazolium (MTT) assay. The cytotoxicity of dilutions (10⁻³, 10⁻⁴, and 10⁻⁵) were evaluated with direct exposure, using the same cell line following ISO 10993-5. SF treatment revealed the highest cell viability among the treatment groups for the dentin-barrier test. In direct cytotoxicity test, SDF and SF treatments exhibited no cytotoxicity at 10⁻⁴ and 10⁻⁵ dilutions. The addition of KI increased cytotoxicity. Ammonia- and water-based silver fluoride treatments, particularly in deep cavities, should be applied with caution.