Chitinase-producing Trichoderma species have been recognized long ago against the phytopathogenic fungi. In this study, we evaluated the production of chitinase enzyme for seventeen isolates of Trichoderma isolated from onion growing districts of Punjab and assessed their bio-efficacy against damping-off in onion. In vitro, these Trichoderma isolates were screened for their antagonistic activity against the damping-off pathogen Fusarium oxysporum f.sp. cepae by dual culture assay. These isolates were also screened for their chitinase enzyme activity; it was found that isolates T5 and T8 are showing higher antagonistic activity on Fusarium oxysporum f.sp. cepae and also produced large amounts of chitinase enzymes in the presence of commercial colloidal chitin. The selected chitinolytic isolates were used in field studies to confirm the feasibility of their biological control efficacy against onion damping-off. In the field experiment, the seed+soil treatment of chitinolytic isolate (T8) showed a critical decrease of damping-off in onion by 88.75% over control.