Following a tendency of studying the potential effects of plant extracts to cancer, this study aimed to evaluate in vitro the cytotoxic activity of Plectranthus neochilus (PN) extracts and its fractions in head and neck squamous cell carcinoma (HNSCC) cell lines and assess their tumor specificity. MTT assay was conducted with two HNSCC cell lines, FaDu (hypopharynx carcinoma) and SCC-25 (tongue carcinoma), one keratinocyte (HaCat) and one fibroblast (L929) cell line. Two PN leaf crude extracts, one ethanolic (E) and one hexanic (H), and their nine fractions were tested. A dose-response curve was performed with hexane PNH fraction and a tumor specificity index (TSI) was calculated. For all cell lines studied, almost all extracts and fractions resulted in cell viability lower than 50%. Hexane and methanol PNH fractions were exceptions, causing a significantly low viability in SCC-25 (17.16 and 34.53%, respectively), but higher than 50% in FaDu, HaCat and L929. The dose-response curve with hexane PNH fraction resulted in a CC 50 of 540.9 µg/mL for FaDu, 550 µg/mL for L929, 762.1 µg/mL for HaCat and 274.2 µg/mL for SCC-25. The TSI L929/FaDu was 1.01, HaCat/FaDu was 1.40, L929/SCC-25 was 2.00 and HaCat/SCC-25 was 2.77. TSIs indicate its specificity for tongue carcinoma cells, when compared to fibroblasts and keratinocytes.