| Summary: | The antifungal and insecticidal effect of the essential oil from <i>Ocimum sanctum</i> L. was evaluated using a model set of harmful organisms hazardous for health and the economy. Toxigenic and plant pathogenic filamentous fungi, including causal agents of human infections, were chosen as exemplary fungal groups—<i>Fusarium verticillioides, Penicillium expansum</i> and <i>Aspergillus flavus</i>. <i>Spodoptera littoralis</i> (African cotton leafworm), <i>Culex quinquefasciatus</i> (Southern house mosquito), the lymphatic filariasis vector and potential Zika virus vector, and the common housefly, <i>Musca domestica</i> were chosen as model insects. Major and minor active substances were detected and quantified using GC/MS analysis. Environmental safety was verified using the non-target useful organism <i>Eisenia fetida</i>. Significant antifungal and insecticidal activity, as well as environmental safety, were confirmed. The essential oil showed the highest efficacy against <i>A. flavus</i> according to MIC50/90, and against <i>S. littoralis</i> larvae according to LD<sub>50/90</sub>. The monoterpenoid alcohol linalool, <i>t-</i>methyl cinnamate, and estragole as phenylpropanoids were detected as effective major components (85.4%). The essential oil from <i>Ocimum sanctum</i> L. was evaluated as universal and significantly efficient, providing a high potential for use in environmentally safe botanical pesticides.
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