Abstract Targeting the use of the second harmonic generation (SHG) as a bioimaging technique to unravel the formation of aggregates, the SHG first hyperpolarizabilities ( ) of assemblies of benzene‐1,3,5‐tricarboxamide derivatives have been evaluated at the density functional theory level. Calculations have revealed that i) the assemblies exhibit SHG responses and the total first hyperpolarizability responses of the aggregates are evolving with their size. The largest aggregation effect is a 18‐times increase for of B4 when going from the monomer to the pentamer, that ii) the intrinsic SHG responses described by the hyper‐Rayleigh Scattering are enhanced in presence of iodine atoms on the phenyl core, that iii) the side chains affect the relative orientation of the dipole moment and first hyperpolarizability vectors, which impacts more the EFISHG quantities than their moduli, and that iv) the radial component to is dominant for the compounds having the largest responses. These results have been obtained using the sequential molecular dynamics then quantum mechanics approach to account for dynamic structural effects on the SHG responses.