The invention relates to fields of pesticides and bacterial inhibitors, which particularly relates to an ophiobolin di-sesquiterpene compound derived from seaweed entophytic fungi, and preparation and application thereof. The structural formula is shown in a formula (I). The preparation method comprise: inoculating Aspergillus ustus cf-42 in a fungus liquid medium to ferment and culture. After purification, the fermentation product is the ophiobolin sesterterpene compound as shown in the formula (I). The ophiobolin di-sesquiterpene compound obtained by the invention can cause a higher death rate of artemia salina sinnaeu which is 78.4% when the compound concentration is 100 microgram/ml. An insecticidal activity experiment suggests that the median lethal concentration is 48.1 microgram/ml. At the meantime, this compound also possesses substantial bacteriostatic activity.