Development of an efficient system for callus induction on different explants of fenugreek. Comparing phenolic, antioxidant and antimicrobial activities of seed and calli of different fenugreek explants. Fenugreek is a well-known medicinal plant for pharmaceutical and nutraceutical properties. For callus induction, three explants were used viz. hypocotyl, root, and cotyledons. The explants were cultured on MS medium supplemented with 2, 4-D, NAA and TDZ in different concentrations (0.5-6 mg/L). Antioxidant activity using DPPH and Folin-ciocalteu assays, and antimicrobial activity of calli and seed extracts were evaluated. Friable callus was successfully induced on all explants and by all PGRs except 2, 4-D which failed to stimulate callus formation on root explant. Root segment was the better explants in callus induction with maximum fresh weight 5.29 g with TDZ at 2 mg/L and highest callus index was 4.3 with TDZ at 0.5 mg/L. Hypocotyl callus showed the highest phenolic content (246.9 mg GAE/g DW) compared to root callus (243.5 mg GAE/g DW), seed (176.2 mg GAE/g DW) and then cotyledons callus (64.9 mg GAE/g DW) with the lowest value. On the other hand, seed exhibited 44.3% antioxidant activity compared to 34.7%, 24.3% and 16.7% recorded by cotyledons, hypocotyl, and root calli, respectively. Regarding antimicrobial activity of studied fenugreek extracts, hypocotyl callus exhibited the maximum zone of inhibition recorded (19 mm) against E. coli. Callus of fenugreek showed potential biological activities over seed-extract.