Two salicylaldehyde-4-hydroxy phenylacetyl acylhydrazone transition metal complexes ZnL2·H2O (I) and [CrL2]NO3·EtOH (II) were synthesized by C15H14N2O3 (HL) with Zn(NO3)2·6H2O and Cr(NO3)3·9H2O. Structures of HL, I and II were characterized by single crystal X-ray diffraction. With the ligand HL belonging to the monoclinic crystal system and the P21/c space group, the coordinating atoms (N and O atoms) in both two complexes I and II exhibited a six-coordinated octahedral geometry centered on the metal ion. Thermal stabilities of the two complexes were analyzed by TG-DTG techniques, which illustrated the maximum thermal decomposition peak temperatures of two complexes were both higher than 170 °C, exhibiting strong thermal stabilities. UV–vis absorption spectroscopy and microcalorimetry were used to determine interaction patterns of I and II with CT-DNA, showing that I and II effectively bound to CT-DNA through the intercalation mode and the interactions processes of I and II with CT-DNA were spontaneous, exothermic entropy reduction reactions. Fluorescence spectroscopy and microcalorimetry were used to determine the interactions of I and II with BSA, presenting that the two complexes can effectively bind to BSA and form non-fluorescent matrix complexes and the interactions processes were spontaneous exothermic and entropy reducing, and each of interaction duration was within 48 min. In addition, the antimicrobial activities of complexes I and II against S. aureus, P. aeruginosa, B. subtilis and E. coli were determined, revealing II exhibited stronger inhibition activity against P. aeruginosa than gentamicin sulfate at 0.5 mg·L-1. Complexes I and II were expected to be used as alternative drugs of gentamicin sulfate in the future.