Five previously undescribed polyacetylene glucosides, namely, four C10- and one C14-acetylenes, together with three known analogues, were isolated from the florets of Carthamus tinctorius L. The structures of these novel compounds were elucidated to be (5R)-5-acetoxy-8,10,12-tetradecatriyne-1-O-β-D-glucopyranoside, (2Z)-decaene-4,6,8-triyne-1-O-β-D-glucopyranoside, (8Z)-1-[(3-O-β-D-glucosyl)-isovaleroyloxy]-8-decaene-4,6-diyne, (8Z)-decaene-1-isovaleroyloxy-4,6-diyne-10-O-β-D-glucopyranoside, and (2E,8E)-decadiene-4,6-diyne-1-O-β-D-glucopyranoside via spectroscopic and chemical methods. All of the isolated compounds were tested for cytotoxicity against cancer cell lines, antibacterial activity, and inhibitory effects on the lipopolysaccharide (LPS)-induced nitric oxide (NO) production. The results showed that (5R)-5-acetoxy-8,10,12-tetradecatriyne-1-O-β-D-glucopyranoside significantly inhibited LPS-induced NO production in RAW264.7 cells in a dose-dependent manner.