In the present work, monoacylglycerol derivatives, i.e., 1-monomyristin, 2-monomyristin, and 2-monopalmitin were successfully prepared from commercially available myristic acid and palmitic acid. The 1-monomyristin compound was prepared through a transesterification reaction between ethyl myristate and 1,2-O-isopropylidene glycerol, which was obtained from the protection of glycerol with acetone, then followed by deprotection using Amberlyst-15. On the other hand, 2-monoacylglycerol derivatives were prepared through enzymatic hydrolysis of triglycerides in the presence of Thermomyces lanuginosa lipase enzymes. The synthesized products were analyzed using fourier transform infrared (FTIR) spectrophotometer, gas or liquid chromatography-mass spectrometer (GC-MS or LC-MS), and proton and carbon nuclear magnetic resonance (¹H- and ¹³C-NMR) spectrometers. It was found that monomyristin showed high antibacterial and antifungal activities, while 2-monopalmitin did not show any activity at all. The 1-monomyristin compound showed higher antibacterial activity against Staphylococcus aureus and Aggregatibacter actinomycetemcomitans and also higher antifungal activity against Candida albicans compared to the positive control. Meanwhile, 2-monomyristin showed high antibacterial activity against Escherichia coli. The effect of the acyl position and carbon chains towards antibacterial and antifungal activities was discussed.