Abstract: Macrolides have been extensively utilized in drug discovery to identify new and highly potent fungistatic compounds. A series of novel ten-, twelve-, and sixteen-membered azolides containing phenoxymethyl and chloromethyl compounds C and E were designed and synthesized based on the macrolide compounds WLD and D16-19, which had previously been investigated by the research group as lead compounds. Their fungistatic activities were evaluated in vitro. The results demonstrated that the majority of the intermediates C and compounds E exhibited inhibitory activities against the five pathogenic fungi tested at a concentration of 50 mg/L. Among these, C5 and C6 showed inhibition rates of 88% and 90% against Alternaria solani, respectively, while C6 exhibited a 94% inhibition rate against Fusarium graminearum. Notably, the addition of structural fragments carbamate and urea did not significantly enhance the compound activity. Furthermore, hexadecyl azalactones C5 and C6 displayed higher inhibitory activity compared to ten- and twelve-membered azalactone C1-C4, indicating that the azalactone ring, as the main structural element of the compounds, had a greater impact on their activity. Compound C5 exhibited an EC₅₀ value of 2.95 mg/L against A. solani, similar to the activity of the control drug pyraclostrobin and superior to the lead compound D16-19 against A. solani (EC₅₀ value of 4.76 mg/L). This suggests that adding phenoxymethyl to hexadecyl azoguanide can enhance the compounds' activity against A. solani. Further investigation is warranted to determine the potential of compound C5 as a novel bacteriostatic lead compound.