The natural 5-azaindoles, marine sponge guitarrin C and D, were observed to exert inhibitory activity against a highly active alkaline phosphatase (ALP) CmAP of the PhoA family from the marine bacterium , with IC values of 8.5 and 110 µM, respectively. The superimposition of CmAP complexes with -nitrophenyl phosphate (NPP), a commonly used chromogenic aryl substrate for ALP, and the inhibitory guitarrins C, D, and the non-inhibitory guitarrins A, B, and E revealed that the presence of a carboxyl group at C6 together with a hydroxyl group at C8 is a prerequisite for the inhibitory effect of 5-azaindoles on ALP activity. The 10-fold more active guitarrin C could compete with NPP for binding sites in the ALP active site due to similarities in size, three-dimensional structure, and the orientation of the COOH group along the phosphate group. However, the inhibition of CmAP and calf intestinal ALP (CIAP) by guitarrin C was observed to occur via a non-competitive mode of action, as evidenced by a twofold decrease in V and an unchanged K. In contrast, the kinetic model with guitarrin D, with an additional OH group at C7, reflected a mixed type of inhibition, with a decrease in both values. The sensitivity of CIAP to guitarrins C and D was shown to be slightly lower than that of CmAP, with IC values of 195 and 230 µM, respectively. Nevertheless, these findings prompted the prediction of complexes of human ALP isoenzymes with guitarrins C and D.