Essential oils (EOs) are complex natural mixture of chemical constituents, having a major component at a relatively high concentration while the minor ones are present in trace amounts. Hence, the need for a simple and efficient isolation technique required to obtain compound that are bioactive antidermatophytic. Therefore, the study aim to isolate bioactive molecules of EO from fruit peels of Nigerian Citrus sinensis (L.) Osbeck with potential antidermatophytic activity against multidrug-resistant tinea capitis dermatophyte. The EO was reaped by hydrodistillation and the bioactive components were examined by thin layer chromatography coupled with direct bioautography assay method. The latter is an assertive technique that targets the isolation of only the active component(s) in a bioassay-guided fashion. The antidermatophytic components were identified with GC-MS as 4- Heptyn-2-ol and Pentane-2, 4- dimethyl. These components (mixture) recorded, are a first-hand report—as present in Citrus sinensis (L.) Osbeck fruit peel EO. Our results support a diagnostic method in the isolation of antidermatophytic components from essential oil with an additional confirmation step.