Crystal structures of two newly synthesized Schiff’s bases with organic fluorine {systematic name: ( E )- N -(4-Fluorophenyl)-1-(5-nitrothiophen-2-yl)methanimine ( 1 ) and ( E )- N -(2-trifluorophenyl)-1- (5-nitrothiophen-2-yl)methanimine ( 2 )} are reported. In this contribution, we performed a qualitative analysis of intermolecular interactions using the Hirshfeld surface and 2D-fingerprint plots to understand the effect of -F and –CF 3 substituents. Compounds 1 and 2 and their respective isomers, -F in the meta and –CF 3 in para position on the phenyl ring, are analyzed and compared. This analysis suggests that three C–H •••O, one C–H •••F and a short S1 •••C4 interactions found to be important for stabilization of crystal structure 1 and three C–H •••O, one C–H •••F, one C–F •••πhalogen bond and π•••πinteractions are found to be vital for stabilization of crystal structure 2 . Further, intermolecular interaction energies of molecular dimers in 1 and 2 are obtained using PIXEL energy analysis. The lattice energies for structures 1, 2 and their isomers are calculated. The energetic analysis suggests that structure 2 is more stable than its para isomer and 1 . Structure 1 and its meta isomer show 1D structural similarity. The quantitative analysis of intermolecular interactions involved in the molecular dimers of 1, 2 and selected dimers of meta isomer of 1 using Bader’s quantum theory of atoms-in-molecules approach. An in vitro evaluation of antimicrobial activities reveals that compounds 1 and 2 exhibit potent and broad spectrum antibacterial activity against selected Gram-positive and Gram-negative bacterial stains.