Background: Spilanthes acmella is used as a remedy in toothache complaints by the tribal people of Western part of Odisha, India. Objective: The objective of this study was to study the growth-arresting activity of an indigenous Acmella essential oil (EO) (S. acmella Murr, Asteraceae) and its isolated component, d-limonene against Trichophyton rubrum (microbial type culture collection 296). Materials and Methods: The EO was extracted from flowers of indigenous S. acmella using Clevenger's apparatus and analyzed by gas chromatography–mass spectrometry (GC-MS). High pressure liquid chromatography (HPLC) was carried out to isolate the major constituent. The isolated fraction was subjected to fourier transform infrared spectroscopy (FTIR) and nuclear magnetic resonance (NMR). The antidermatophytic activity was screened for using “disc diffusion” and “slant dilution” method followed by optical, scanning electron microscopy (SEM), and transmission electron microscopy (TEM) studies. The molecular dockings were made between d-limonene with cell wall synthesis-related key enzymes (14 methyl deaminase and monooxygenase). Results: The GC-MS analysis EO had inferred the presence of 7 number of major (≥2%) components. The component with highest peak area (%) was found to be 41.02. The HPLC-isolated fraction was identified as d-limonene (1,8 p-Mentha-diene) by FTIR and NMR. Qualitative and quantitative assays had suggested the growth inhibitory activity of Acmella EO and its component. Shrinkage, evacuation, cell wall puncture, and leakage of cellular constituents by the activity of Acmella oil and d-limonene were evidenced from optical, SEM, and TEM studies. The computer simulation had predicted the binding strengths of d-limonene and fluconazole with dermatophyte cell wall enzymes. Conclusion: There could have been synergistic action of all or some of compounds present in indigenous Acmella EO.