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The present investigation reports the influence of ethylene inhibitor and elicitors on enhanced biosynthesis of colchicine and thiocolchicoside contents in cell suspension cultures of Gloriosa superba L. Elicitation is one of the approaches used for enhanced commercial production of secondary metabolites from plant cell culture system. G. superba is a rich source of colchicine and thiocolchicoside contents that are being used for the treatment of cancer. Callus was obtained from rhizomes of G. superba on MS medium containing 2.0 mg L−1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.5 mg L−1 α-naphthalene acetic acid (NAA) and cell suspension cultures were initiated. The influence of elicitors [salicylic acid (SA), yeast extract (YE), and casein hydrolysate (CH)] as well as ethylene inhibitor [silver nitrate (AgNO3)] on biosynthesis of colchicine and thiocolchicoside contents was studied by exposure of cell suspension cultures with different concentrations for 15 and 30 days period. Among four elicitors tested, CH exhibited the maximum level of colchicine production [8.290 mg g−1 dry weight (DW)] at 300 mg L−1 concentration over a period of 15 days exposure, compared to the control whereas, SA at 27.624 mg L−1 concentration showed the enhanced colchicine production rate (8.149 mg g−1 DW) after 30 days exposure time when compared to non-elicited control cultures. Results show that CH and SA treated cells exhibited 8-fold higher level of colchicine biosynthesis over control. The maximum production of thiocolchicoside content in cell suspension culture obtained was 4.550 mg g−1 DW and 1.530 mg g−1 DW with 200 and 300 mg L−1 AgNO3 treatment at 15 and 30 days period, respectively. The colchicine content level was significantly increased when compare to the thiocolchicoside content in cell suspension culture. The accumulation of colchicine and thiocolchicoside contents was influenced by exposure time and doses of elicitors. Results clearly show that the elicitation experiment could enhance the biosynthesis of colchicine and thiocolchicoside contents level in cell suspension cultures of G. superba and the present protocol may be used for commercial supply of these important secondary metabolites in the future for therapeutic applications.