•Novel 16-membered azalides and azalactams were synthesized from cyclododecanone.•This is the first time to introduce 2-methylene into macrolide skeleton.•α-Exomethylene and p-chlorobenzoyl groups were found to enhance antifungal activity.Novel macrocyclic molecules comprising sulfonyl and acyl moiety at the position N-12a of 16-membered azalides (6a–n) and azalactams (10a–r) scaffold were synthesized from cyclododecanone 1 as starting material via 5 steps and 4 steps, respectively. The antifungal activity of these compounds against Sclerotinia sclerotiorum, Pyricularia oryzae, Botrytis cinerea, Rhizoctonia solani and Phytophthora capsici were evaluated and found that compounds possessing α-exomethylene (6c, 6d, 6e and 6g) showed antifungal activity comparable to commercial fungicide Chlorothalonil against P. oryzae and compounds possessing p-chlorobenzoyl exhibited enhanced antifungal activity than those with other substituents against S. sclerotiorum, P. oryzae, and B. cinerea. These findings suggested that the α-exomethylene and p-chlorobenzoyl may be two potential pharmacological active groups with antifungal activities.Novel 16-membered azalides and azalactams derivatives were synthesized and shown to have moderate to high inhibitory activity against five important plant pathogenic fungi.