Lagunamides A (1) and B (2) are new cyclic depsipeptides isolated from the marine cyanobacterium Lyngbya majuscula obtained from Pulau Hantu Besar, Singapore. The planar structural characterization of these molecules was achieved by extensive spectroscopic analysis, including 2D NMR experiments. In addition to Marfey’s method and 3JH−H coupling constant values, a modified method based on Mosher’s reagents and analysis using LC-MS was deployed for the determination of the absolute configuration. Lagunamides A and B displayed significant antimalarial properties, with IC₅₀ values of 0.19 and 0.91 μM, respectively, when tested against Plasmodium falciparum. Lagunamides A and B also possessed potent cytotoxic activity against P388 murine leukemia cell lines, with IC₅₀ values of 6.4 and 20.5 nM, respectively. Furthermore, these cyanobacterial compounds exhibited moderate antiswarming activities when tested against Pseudomonas aeruginosa PA01.