Avermectin produced by Streptomyces avermitilis is an anti-nematodal agent against the pine wood nematode Bursaphelenchus xylophilus. However, its potential usage is limited by its poor water solubility. For this reason, continuous efforts are underway to produce new derivatives that are more water soluble. Here, the enzymatic glycosylation of avermectin was catalyzed by uridine diphosphate (UDP)-glycosyltransferase from Bacillus licheniformis with various UDP sugars. As a result, the following four avermectin B1a glycosides were produced: avermectin B1a 4″-β-D-glucoside, avermectin B1a 4″-β-D-galactoside, avermectin B1a 4″-β-L-fucoside, and avermectin B1a 4″-β-2-deoxy-D-glucoside. The avermectin B1a glycosides were structurally analyzed based on HR-ESI MS and 1D and 2D nuclear magnetic resonance spectra, and the anti-nematodal effect of avermectin B1a 4″-β-D-glucoside was found to exhibit the highest activity (IC50 = 0.23 μM), which was approximately 32 times greater than that of avermectin B1a (IC50 = 7.30 μM), followed by avermectin B1a 4″-β-2-deoxy-D-glucoside (IC50 = 0.69 μM), avermectin B1a 4″-β-L-fucoside (IC50 = 0.89 μM), and avermectin B1a 4″-β-D-galactoside (IC50 = 1.07 μM). These results show that glycosylation of avermectin B1a effectively enhances its in vitro anti-nematodal activity and that avermectin glycosides can be further applied for treating infestations of the pine wood nematode B. xylophilus.