Two new harziane diterpenes (
1–
2), five undescribed cyclonerane sesquiterpenes (
3–
7), and three known compounds, 11-cycloneren-3, 7, 10-triol (
8), harziandione (
9), and dehydroacetic acid (
10), were isolated from
Trichoderma hamatum
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Two new harziane diterpenes (
1–
2), five undescribed cyclonerane sesquiterpenes (
3–
7), and three known compounds, 11-cycloneren-3, 7, 10-triol (
8), harziandione (
9), and dehydroacetic acid (
10), were isolated from
Trichoderma hamatum b-3. Their structures were elucidated via comprehensive inspection of spectral evidence in HRESIMS and 1D and 2D NMR, and the absolute configuration of
1–
8 was confirmed by NMR, ECD calculation, as well as Mosher’s method. In vitro fungicidal activity showed that some compounds showed great inhibitory activity against pathogenic fungi, including
Fusarium graminearum,
Sclerotinia sclerotiorum,
Botrytis cinerea, and
Rhizoctonia solani, among which compound
10 showed 100% inhibition of
S. sclerotiorum and
B. cinerea. The in vivo activity test showed that compound
10 was 65.8% effective against
B. cinerea and compound
10 can be used as a lead compound for the development of biopesticides that inhibit
B. cinerea. This study elucidated the bioactivity of secondary metabolites of
T. hamatum and indicated the direction for the subsequent development of the biological control activity of
T. hamatum.
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