A novel series of six mixed transition metal and alkaline earth metal complexes of the general formulation [MM/(C4H4O6)2.XH2O] (where M/=Ba and M= Mn, Fe, Co, Ni, Cu, and Zn ) are synthesized by using bidentate tartarate ligand and are characterized by different analytical techniques such as elemental analysis, TGA, FTIR, XRD, magnetic susceptibility study, UV-visible spectroscopy etc. To check the performance all synthesized mixed metal complexes (sample B1 to B6) were then tested for in-vitro anti-biofilm activity against some fresh bacterial cultures namely of Pseudomonas aeruginosa ATCC-27853, Escherichia coli ATCC-25922, Staphylococcus aureus ATCC-25923, Klebsiella pneumoniae (Lab culture), and Proteus vulgaris (Lab culture). As a result most challenging and important observation was that Minimum Bactericidal Concentration (MBC) of these complexes was found slightly more than Minimum Inhibitary Concentration (MIC). It has been observed that the Biofilm Inhibition Concentration (BIC) levels of all the complexes are at higher side as compared to MIC but lower to MBC. Bio assays of all the complexes show a greater biofilm inhibition effect in comparison to the individual tartarate ligand which reports that after coordination the anti-biofilm activity of complexes is becoming superior and enhanced. Distinctly B2 and B6 were found more active in obstructing biofilm of test bacterial cultures, which demonstrates they are very agile or energetic in inhibition, for minimizing dwelling of bacteria in hospital instruments, in dockyard for preventing fouling of the surface of ship and depending on animal and human toxicity, which can be used to control infections caused by such microorganisms.